IL-17A binding peptides and medical uses thereof

ABSTRACT

The invention relates to IL-17A binding peptides, inhibitors of the interaction of IL-17A with the receptor IL-17RA, and to bioconjugates, dimers, pharmaceutical compositions and medical use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims the benefit of PCTApplication No. PCT/EP2019/070265, filed Jul. 26, 2019, which claimsbenefit of European Patent Application No. 18186029.7, filed Jul. 27,2018, both of which are incorporated by reference herein in theirentirety.

REFERENCE TO SEQUENCING LISTING FILED ON EFS-WEB

The instant application contains a sequence listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 17, 2021, isnamed “Sequence_Listing.txt” and is 50,167 bytes in size.

The present invention relates to IL-17A binding peptides, inhibitors ofthe binding of IL-17A to the IL-17AR receptor and thereby of theformation of the complex IL-17A/IL-17RA/IL17RC. Object of the inventionare also bioconjugates and dimers comprising the above peptides,pharmaceutical compositions and medical uses of the above peptides,bioconjugates and dimers.

BACKGROUND OF THE INVENTION

Human cytotoxic T lymphocyte-associated antigen 8 (CTLA8), named alsoInterleukin 17 (IL-17), was first cloned in 1993. The first biologicalactivity described for human IL-17 was the induction of the productionof Interleukin 6 (IL-6) and Interleukin 8 (IL-8) from rheumatoidarthritis synoviocytes. This finding suggested a role for IL-17 ininflammation through IL-6, and in neutrophil recruitment through IL-8 XuS et al, Cell Mol Immunol. 2010, 164-74; Murcia R Y et al, PLOS ONE2016, 11(5): e0154755doi:10.1371/journal.pone.0154755).

It was later found that this molecule, later named IL-17A, is part of alarger family which includes five additional members, namely IL-17B toF. IL-17F shares the greatest homology (about 56%) with the firstdiscovered IL-17, IL-17A, while IL-17E displays the lowest sequenceconservation (about 16%). The members of IL-17 family exert theirfunctions binding to their receptors in the form of homodimers, with theexception of IL-17A and IL-17F which may also act as heterodimers(Goepfert A et al, Sci Rep. 2017, 7 (1), 8906).

The most widely investigated cytokine of this family, IL-17A, plays anessential role in host defense against microbial infections and it isconsidered the major driver for a number of inflammatory and autoimmuneconditions. Pathological production of IL-17A leads to excessiveinflammation and evident tissue damage.

In particular, through induction of a variety of molecules includingcytokines, chemokines, acute phase proteins, anti-microbial peptides,mucins, and matrix metalloproteinases, IL-17A can amplify cascades ofevents that lead to neutrophil recruitment, inflammation and hostdefense. Although IL-17A is the signature cytokine produced by T helper17 (Th17) cells, IL-17A, as other IL-17 family cytokines, has multiplesources ranging from immune cells to nonimmune cells.

IL-17B, IL-17C, and IL-17D are also considered pro-inflammatorycytokines but their role is not fully known. IL-17E, also known asIL-25, has the lowest homology and is involved in Th2 cell responsesagainst parasites and allergy. CCL20 drives the recruitment of Th17 anddendritic cells to the inflammatory site. In turn, Th17 cells areactivated thus producing inflammatory mediators and leading to chronicinflammation.

The cytokines belonging to IL-17 family signal via their relatedreceptors and activate downstream pathways that include NFκB, MAPKs andC/EBPs to induce the expression of antimicrobial peptides, cytokines andchemokines. The proximal adaptor Act1 is a common mediator during thesignaling of all IL-17 cytokines, and it is involved in IL-17-mediatedhost defense and in IL-17-driven autoimmune conditions.

The IL-17 receptor family consists of five members, IL-17RA, RB, RC, RDand RE, all of which, like their ligands, share sequence homology.

IL-17RA is ubiquitously expressed on a wide range of tissues and celltypes and binds to IL-17-A, C, E and F. Receptor signaling occursthrough heterodimeric receptors formed of a common IL17-RA subunit and asecond subunit that depends on the ligand and regulates signalingspecificity. Upon stimulation with the ligand, IL-17RA forms aheterodimeric receptor complex with IL-17RB (for IL-17E), IL-17RC (forIL-17A and IL-17F) or IL-17RE (for IL-17C). It is proposed that thebinding of ligand to the first IL-17RA receptor subunit promotes thesecond binding event, thus inducing the formation of a heterodimericreceptor complex. In particular, the signal of IL-17A and IL-17F ismediated by the complex between IL-17RA and IL-17RC. IL-17F binds toIL-17RA with about 100 to 1000 times lower affinity than IL-17A, whilethe binding affinity for IL-17RC is comparable between the two cytokines(Onishi R M et al, Immunology 2010, 129(3), 311-321; Gu C et al,Cytokine 2013, 64(2), 477-485).

IL-17A in humans plays a pivotal role in various autoimmune andinflammatory conditions, such as rheumatoid arthritis, multiplesclerosis, psoriasis, Crohn's disease, systemic lupus erythematosus,asthma, Behçet's disease, and hyper IgE syndrome (Fujino S et al Gut,2003, 52, 65-70; Shabgah A G et al, Postepy Dermatol Allergol 2014, 31(4), 256-261). Furthermore, IL-17A blockade has shown preclinical andclinical efficacy in ankylosing spondylitis and rheumatoid arthritis(Liu S et al, Nature-Scientific Reports 2016, doi:10.1038/srep26071;Lubberts E et al, Arthritis Rheum 2004, 50, 650-659).

Also, IL-17A has been shown to be involved in ocular diseases, and inparticular in the pathogenesis of ocular surface and corneal diseases,such as dry eye disease (DED), viral and bacterial keratitis. Antibodiesdeveloped to neutralize IL-17A have shown promising results in reducingthe severity of these diseases (Garbutcheon-Singh K B et al, Curr EyeRes, 2018, D01:10.1080/02713683.2018.1519834).

DED is an inflammatory and autoimmune ophthalmic disease of the tearsystem and ocular surface that results in discomfort, visual disturbanceand tear film impairment. DED is one of the most common cause ofacquired visual impairment in adult population (Stem M E et al, MucosalImmunol 2010, 3(5), 425-442; Stevenson W et al, Arch Ophthalmol 2012,130(1), 90-100, Parul Singh, Parul Singh 2012, Hah, Chung et al. 2017).The disease involves an immune and inflammatory process that affects theocular surface and, in severe cases, may lead to blindness. Currenttreatment of DED is mainly symptomatic, consisting in ocular lubricantsand non-specific anti-inflammatory agents such as corticosteroids,cyclosporine A, and tacrolimus. IL-17A is associated with damage to thecorneal epithelial barrier function, which is the most sight threateningcomplication of DED. An increase in Th17 cells is reported in DEDpatient ocular tissues that induces an increase in IL-6, TGF-β, IL-23and IL-17A concentrations on the ocular surface, as well as increasedconcentration of IL-17 in tears, and number of Th17 cells on the ocularsurface of experimental DED models. In addition, it has also beendemonstrated that in vivo neutralization of IL-17A results in a markedlyattenuated induction and severity of disease (De Paiva C S et al,Mucosal Immunol. 2009; 2(3): 243-253, Chauhan et al, Mucosal Immunol,2009, 2(3), 243-253; Chauhan S K et al, Mucosal Immunol 2009, 2(4),375-376; Chauhan, El Annan et al. 2009, De Paiva, Chotikavanich et al.2009, de Paiva, Huang et al. 2014, Subbarayal, Chauhan et al. 2016).

IL-17A has also been implicated in psoriasis. Psoriasis is a chronicinflammatory disease. It manifests itself as dry, raised, red skinlesions (plaques) covered with silvery scales. Numerous clinicalphenotypes exist (i.e. plaque, guttate, pustular, inverse), with diseaseseverity ranging from a few scattered plaques to extensive body surfaceinvolvement. Individuals with psoriasis have an increased risk ofdeveloping other chronic and serious diseases such as psoriaticarthritis, metabolic syndrome, cardiovascular diseases and depression. Acentral role of IL-17A in the pathophysiology of psoriasis has beendemonstrated (Zeichner J A et al J Clin Aesthet Dermatol 2016, 9 (6Suppl 1), S3-S6). In psoriasis, expression of IL-17 mRNA is higher inlesional compared with nonlesional skin. Additionally, IL-17A levels aresignificantly correlated to disease severity (Arican O et al. MediatorsInflamm 2005, 2005, 273-279, Takahashi K et al. Clin Exp Dermatol 2010,35, 645-649). Blockade of IL-17A has been shown to reduce keratinocytehyperproliferation, T-cell infiltration into the dermis, and mRNAexpression of key disease-propagating genes (Krueger J G et al, JAllergy Clin Immunol 2012, 130, 145-154).

Anti-IL-17A antibodies secukinumab (AlN457, Consentyx™), a fully humanIgG1k anti-IL-17A mAb, and ixekizumab (LY2439821), a humanized IgG4antibody, were tested in clinical trials in psoriasis and approved forthe treatment of moderate to severe psoriasis. During clinical trials,both antibodies were able to reduce the severity of the disease by atleast 75% (PASI75) in 80% of the patient population. Moreover, currentadvanced clinical trials have shown promising results also for treatmentof ankylosing spondylitis and psoriatic arthritis. Other antibodies arenow in clinical trials for the same indications.

The above evidence strongly supports the development of molecules ableto target IL-17A and inhibit its signaling for the treatment of theabove pathologies. Until recently, targeting of IL17-A and its receptorshas remained the domain of antibodies. The reason for the preference forantibodies is that cytokines, as IL-17A, constitute a protein-proteininteraction (PPI) target difficult to target, so far consideredundruggable. In fact, PPI interfaces are generally flat and lack deepsubpockets and grooves that are usually necessary to bind SmallMolecular Weight (SMW) molecules. Despite this, in recent years newresearch identified a few SMW IL-17A antagonists able to bind IL-17A andprevent its interaction with IL-17RA receptor, and thus inhibit IL-17Apathway activation (Espada A et al. J Med Chem 2016, 59(5), 2255-2260;Ting J P et al PLoS One 2018, 13(1), e0190850; Liu S et al, Scientificreports, 6:26071, doi: 10.1038/srep26071). Among these, a peptide ofsequence IHVTIPADLWDWINK (SEQ ID NO: 1), which was named High AffinityPeptide (HAP) has been described (Liu S et al, Scientific reports,6:26071, doi: 10.1038/srep26071).

However, there is still the need of identifying and characterizing newmolecules able to bind with high affinity to IL-17A and prevent IL-17Apathway activation for the development of new therapeutic options forthe treatment of disorders involving IL-17A/IL-17RA axis.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found peptides able to bind to aspecific site on the N-terminal portion of IL-17A and thereby to inhibitthe interaction with IL-17RA and the formation of theIL-17A71L-17RA/IL17RC complex.

The inhibition of said interaction blocks the IL-17A signaling pathway.The peptides according to the invention are therefore able to reduceTH17-related inflammation and the subsequent damage observed ininflammatory and autoimmune diseases where these cells and IL-17A exerta pivotal role.

As will be shown in the experimental section, the peptides of theinvention have physical-chemical properties that make them particularlysuitable for topical treatment of ophthalmic and dermatologicaldiseases.

Said peptides are useful to design innovative and specific topicaltreatment of diseases dependent on an excessive production or activityof IL-17A.

Objects of the present invention are peptides able to inhibit IL-17Abinding to IL-17RA, dimers and bioconjugates, pharmaceuticalcompositions comprising said peptides, dimers or bioconjugates and theuse of the above in the prevention and/or treatment of autoimmune andinflammatory diseases depending on IL-17A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows some dose-response curves obtained in the IL-17RA-IL17RCdimerization assay described in Example 5 of with representative IL-17Abinding peptides according to the invention. The percentage ofinhibition of IL-17RA/IL17RC dimerization induced by IL-17A obtained bytreatment with different peptides is reported.

FIG. 2 shows the activity of the IL-17A binding peptide of SEQ ID NO:18on secretion of IL6, IL-17A and IL23 by human differentiated andactivated TH17 cells, measured as described in Example 6. Theconcentration of the different cytokines in the supernatant of TH17cells with or without (control) peptide of SEQ ID NO:18 is reported.

FIG. 3 shows the activity of the IL-17A binding peptide of SEQ ID NO:18on secretion of Metalloproteinase 3 by human differentiated andactivated TH17 cells, measured as described in Example 6. Theconcentration of Metalloproteinase 3 in the supernatant of TH17 cellswith or without (CONTROL) peptide of SEQ ID NO:18 is reported.

FIG. 4 shows viability of human TH17 cells after 24 h, in the presenceand absence (control) of the peptide of SEQ ID NO: 18, as described inExample 6.

FIG. 5 shows the binding site on IL-17A of the IL-17A peptides accordingto the invention.

FIG. 6 shows the sensograms obtained in the experiment of Example 9 withthe peptide of SEQ ID NO: 2 (upper panel) or SEQ ID NO: 1 (lower panel).

FIG. 7 shows the value of the affinity constant at differentconcentrations of the peptide of SEQ ID NO: 2 (upper panel) or SEQ IDNO: 1 (lower panel), obtained in the experiment of Example 9.

FIGS. 8A-8B show the level of expression of IL-8 measured by real-timePCR as described in Example 10 in HCEC cells not treated (vehicle) ortreated with the peptide of SEQ ID NO: 2 (FIG. 8A) or with the peptideHAP (SEQ ID NO: 1) (FIG. 8B). The error bars represent the standarddeviation. T-test was performed as statistical analysis. P-value *<0.05,**<0.005, ***<0,0005.

FIGS. 9A-9B show the amount of IL-6 measured by real-time PCR asdescribed in Example 10 in HCEC cells not treated (vehicle) or treatedwith the peptide of SEQ ID NO: 2 (FIG. 9A) or with the peptide HAP (SEQID NO: 1) (FIG. 9B). The error bars represent the standard deviation.T-test was performed as statistical analysis. P-value *<0.05, **<0.005,***<0,0005.

FIGS. 10A-10B show the amount of TNF-α measured by real-time PCR asdescribed in Example 10, in HCEC cells not treated (vehicle) or treatedwith the peptide of SEQ ID NO: 2 (FIG. 10A) or with the peptide HAP (SEQID NO: 1) (FIG. 10B). The error bars represent the standard deviation.T-test was performed as statistical analysis. P-value *<0.05, **<0.005,***<0,0005.

DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those persons skilled in the art to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference;thus, the inclusion of such definitions herein should not be construedto represent a substantial difference over what is generally understoodin the art.

The term “pharmaceutically acceptable excipient” refer to substancesother than the active pharmaceutical ingredient (API) that have beenappropriately evaluated for safety and are intentionally included in adrug delivery system. Pharmaceutically acceptable excipients are wellknown in the prior art and are disclosed, for example in the Handbook ofPharmaceutical Excipients, seventh edition 2013, which is included herefor reference (Rowe, Sheskey et al. 2012).

Excipients are normally classified according to the function that theyhave in the final pharmaceutical composition. Preferably, suitableexcipients according to the present invention are for example diluent,adsorbent, glidant, binder, lubricant, surfactant, disintegrating,preservatives, antioxidant or mixtures thereof.

The terms “approximately” and “about” herein refers to the range of theexperimental error, which may occur in a measurement.

The terms “comprising”, “having”, “including” and “containing” are to beconstrued open-ended terms (i.e. meaning “including, but not limitedto”) and are to be considered as providing support also for terms as“consist essentially of”, “consisting essentially of”, “consist of” or“consisting of”.

The terms “consist essentially of”, “consisting essentially of” are tobe construed as semi-closed terms, meaning that no other ingredientswhich materially affects the basic and novel characteristics of theinvention are included (optional excipients may thus be included).

The terms “consists of”, “consisting of” are to be construed as closedterms.

The term “bioconjugate” as used herein refers to a conjugate formed by astable covalent link between different molecules, preferably twomolecules, optionally linked by means of a spacer, at least one of whichis a biomolecule.

The term “biomolecule” as used herein refers to molecules of biologicalorigin. The term includes macromolecules, such as carbohydrates, lipidsand proteins or small natural products. For the purpose of the presentinvention “biomolecules” are preferably selected from ascorbic acid,capric acid, capronic acid, N-Acetyl-Glucosamine (also referred to asNAG), N-Acetylmuramic acid (also referred to as NAM), hyaluronic acid,alginic acid, chitin, (GaINAc)₂, GaI-alpha1,3-GaINAc or trigalacturonicacid

The definition “conservative substitution” herein refers to aconservative replacement (also called a conservative mutation or aconservative substitution) is an amino acid replacement that changes agiven amino acid to a different amino acid with similar biochemicalproperties (Simon French 1983).

DESCRIPTION OF THE INVENTION

The present inventors have identified a number of peptides that are ableto bind with high affinity to IL-17A and inhibit its interaction withthe ILRA receptor. These compounds act as inhibitors of IL-17Asignaling.

Accordingly, a first object of the present invention is a peptide, ableto inhibit IL-17A binding to ILRA, having the aminoacid sequence ofeither:

formula (I):

(I) (SEQ ID NO: 258) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅

wherein, independently from each other:

X₁ is I, V, D, K, W, A, G, L or P;

X₂ is H, M, K, N, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, H, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₆ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q or it is absent;

X₁₁ is D, E or N or it is absent;

X₁₂ is W, F, V, H or Y or it is absent;

X₁₃ is I, V, F, E, K, A, G, L, P or Y or its absent;

X₁₄ is N, R, E, F, Q or D or it is absent;

X₁₅ is K, R, E, F, V, W, H or D or it is absent;

with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ IDNO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereofIHVTIPADLWDWIN (SEQ ID NO: 259), IHVTIPADLWDWI (SEQ ID NO: 260), orIHVTIPADLWDW (SEQ ID NO: 261);

or formula (II):

(II) a_(n)-DLSAVCWAFPWDPECH-b_(n), (an-SEQ ID NO: 41-bn′

wherein, independently from each other:

a, b are selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S,T, W, Y or V;

n=integer from 0 to 3;

n′=integer from 0 to 3.

Preferably, in formula (II) each of the aminoacids different from a or bcan be replaced with a conservative substitution.

A preferred peptide of formula (II) is DLSAVCWAFPWDPECH (SEQ ID NO: 41).

Particularly preferred are the peptides of formula (I).

According to a preferred embodiment, in said peptide of formula (I):

X₁ is I, V, D, K or W;

X₂ is H, M, K or N;

X₃ is V or F;

X₄ is T, Q, H, V or Y;

X₅ is I or F;

X₆ is P or G;

X₇ is A or Q;

X₈ is D, E, N;

X₉ is L, V, F or W;

X₁₀ is W, Y, F or it is absent;

X₁₁ is D, E, N or it is absent;

X₁₂ is W, F, V or it is absent;

X₁₃ is I, V, F or it is absent;

X₁₄ is N, R, E, F or it is absent;

X₁₅ is K, R, E, F, V, W or it is absent;

with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ IDNO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereofIHVTIPADLWDWIN (SEQ ID NO: 259), IHVTIPADLWDWI (SEQ ID NO: 260), orIHVTIPADLWDW (SEQ ID NO: 261).

According to an alternative preferred embodiment, in said peptide offormula (I):

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q or it is absent;

X₁₁ is D, E or N or it is absent;

X₁₂ is W, F, H or Y or it is absent;

X₁₃ is I, V, F, E, K, A, G, L, P or Y or its absent;

X₁₄ is N, R, E, Q or D or it is absent;

X₁₅ is K, R, E, V, W, H or D or it is absent;

with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ IDNO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereofIHVTIPADLWDWIN (SEQ ID NO:259), IHVTIPADLWDWI (SEQ ID NO:260), orIHVTIPADLWDW (SEQ ID NO:261).

Preferably, in the above peptide of formula (I) all aminoacids X₁ to X₁₅are present and the above described peptides of formula (I) have a 15aminoacid sequence. Preferably, in the peptides of formula (I) having asequence of less than 15 aminoacids, when one aminoacid is absent, alsoall aminoacids at the N terminal of said aminoacid are absent. Forexample, if aminoacid X₁₀ is absent, also aminoacids X₁₁ to X₁₅ areabsent.

Particularly preferred peptides of formula (I) are those wherein X₁ isV.

A particular preferred peptide having X₁ as V is the peptide of SEQ IDNO: 2.

As will be demonstrated in the experimental section, the presentinventors have found that the peptide of SEQ ID NO: 2, where the firstaminoacid Isoleucin in the peptide HAP of SEQ ID NO:1 has been replacedby a Valine, surprisingly shows strikingly different functional andchemical-physical properties as well as improved tolerability comparedto HAP.

In particular, as demonstrated in the Examples, the peptide showsimproved chemical-physical properties, that are particularlyadvantageous for topical and ophtalmic use, a better permeability and anincreased affinity for IL-17A compared to the corresponding peptide ofSEQ ID NO: 1. Also, the inventors have found that the peptide HAP,independently from its activity on IL-17A signaling, exerts a directtoxicity effect on corneal cells by inducing the expression ofinflammatory cytokines. Surprisingly, the peptide of SEQ ID NO: 2 doesnot show this effect and therefore is characterized by an improvedtolerability compared to HAP.

Preferably, in the above peptide of formula (I):

X₁ is I, V or L;

X₂ is H, M, R, K or E;

X₃ is V, F or I;

X₄ is T, Q, S, Y or N;

X₅ is I, F or V;

X₆ is P;

X₇ is A, Q, or L;

X₉ is D, E, or Q;

X₉ is L, W, F, V or I;

X₁₀ is W, Y or F;

X₁₁ is D, E or N;

X₁₂ is W or F;

X₁₃ is I, V, F or L;

X₁₄ is N, R, Q or E;

X₁₅ is K, R, H or E;

-   -   with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ        ID NO: 1);

or

X₁ is I or V;

X₂ is H, M or R,

X₃ is V or F;

X₄ is T or Q;

X₅ is I, F or V;

X₆ is P or G;

X₇ is A or Q;

X₈ D or E;

X₉ is L;

X₁₀ is W or Y;

X₁₁ is D or E;

X₁₂ is W;

X₁₃ is I or V;

X₁₄ is N, R or E;

X₁₅ is K, R or E;

with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO:1);

or

X₁ is I or V;

X₂ is H or M,

X₃ is V;

X₄ is T;

X₅ is I;

X₆ is P;

X₇ is A;

X₈ is D;

X₉ is L, W, F, V or I;

X₁₀ is W or Y;

X₁₁ is D or E;

X₁₂ is W;

X₁₃ is I or V;

X₁₄ is N, R or E;

X₁₅ is K, R or E;

with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO:1);

or

X₁ is I, V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₈ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₈ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, O, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L or P;

X₂ is M, K, N, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, Nor Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L or P;

X₂ is H, M, K, N, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or O;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is V, A, G, L or P;

X₂ is M, K, N, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, O, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, Nor Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₅ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is Y, F or Q;

X₁₁ is E or N;

X₁₂ is W, F, H or Y;

X₁₃ is V, F, E, K, A, G, L, P or Y;

X₁₄ is N, R, E, Q or D;

X₁₅ is K, R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is R, E, Q or D;

X₁₅ is R, E, V, W, H or D;

or

X₁ is V, A, G, L, or P;

X₂ is H, M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, Nor Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is R, E, Q or D;

X₁₅ is R, E, V, W, H or D;

or

X₁ is I, V, A, G, L, or P;

X₂ is M, K, R, E, Q, W or Y;

X₃ is V, F, A, G, L, P, I, Y or W;

X₄ is T, Q, S, N or Y;

X₅ is I, F, A, G, L, P, V or Y;

X₆ is P, G, A, L, V, I or N;

X₇ is A, Q, G, L, P, V, I, N or E;

X₈ is D, E, N, Q or Y;

X₉ is L, V, F, W, A, G, P, I or H;

X₁₀ is W, Y, F or Q;

X₁₁ is D, E or N;

X₁₂ is W, F, H or Y;

X₁₃ is I, V, F, E, K, A, G, L, P or Y;

X₁₄ is R, E, Q or D;

X₁₅ is R, E, V, W, H or D.

Particularly preferred peptides of formula (I) among those describedabove are those wherein

X₁ is V.

As will be demonstrated in the experimental section, when the firstaminoacid in the peptide of formula (I) is a valine, the peptidesurprisingly shows chemical-physical properties particularlyadvantageous for topical and ophthalmic use and an increased affinityfor IL-17A.

Preferably, in the above peptide of formula (I) all aminoacids X₁ to X₁₅are present. Preferred individual peptides of formula (I) according tothe present invention are listed in Table 1 below.

TABLE 1 SEQ ID NO: Sequence SEQ ID NO: 2 VHVTIPADLWDWINK SEQ ID NO: 3IMVTIPADLWDWINK SEQ ID NO: 4 VMVTIPADLWDWINK SEQ ID NO: 5IHVTIPAELWDWINK SEQ ID NO: 6 IHVTIPADLYDWINK SEQ ID NO: 7IHVTIPADLYEWINK SEQ ID NO: 8 IHVTIPADLWDWVNK SEQ ID NO: 9IHVTIPADLWDWIRK SEQ ID NO: 10 IHVTIPADLWDWINR SEQ ID NO: 11IHVTIPADLWDWIEK SEQ ID NO: 12 IHVTIPADLWDWINE SEQ ID NO: 13IHVTIPADLYEWINK SEQ ID NO: 14 IHVTIPADLWDWVRR SEQ ID NO: 15IHVTIPADLWDWVEE SEQ ID NO: 16 VMVTIPADLYEWINK SEQ ID NO: 17VMVTIPADLYEWIRR SEQ ID NO: 18 VMVTIPADLYEWIEE SEQ ID NO: 42IMVTIPADLYEWIEE SEQ ID NO: 111 VHVTIPAELWEWVRR SEQ ID NO: 112VHFTIPADLWEWVRR SEQ ID NO: 113 VHVQIPADLWEWVRR SEQ ID NO: 114VHVTFPADLWEWVRR SEQ ID NO: 115 VHVTIPQDLWEWVRR SEQ ID NO: 116VHVTIPANLWEWVRR SEQ ID NO: 117 VHVTIPADFWEWVRR SEQ ID NO: 118VHVTIPADLYEWVRR SEQ ID NO: 119 VHVTIPADLWNWVRR SEQ ID NO: 120VHVTIPADLWEFVRR SEQ ID NO: 121 VHVTIPADLWEWFRR SEQ ID NO: 122VHVYIPAELWEWVRR SEQ ID NO: 123 VHVTIPAEWWEWVRR SEQ ID NO: 124VHFTFPQDLWEWVRR SEQ ID NO: 125 VHFTFPQDFWEWVRR SEQ ID NO: 126VHFTIPQDLYEWVRR SEQ ID NO: 127 VHFTFPQDLWNWVRR SEQ ID NO: 128VHFTFPQDLWEFVRR SEQ ID NO: 129 VHFTFPQDLWEWFRR SEQ ID NO: 130VHFQFPADLWEWVRR SEQ ID NO: 131 VHFQFPADFWEWVRR SEQ ID NO: 132VHFQFPADLYEWVRR SEQ ID NO: 133 VHFQFPADLWNWVRR SEQ ID NO: 134VHFQFPADLWEFVRR SEQ ID NO: 135 VHFQFPADLWEWFRR SEQ ID NO: 136VHFQFPQDWWEWVRR SEQ ID NO: 137 VHFQIPQDWWEWVRR SEQ ID NO: 138VHFQFPQDWYEWVRR SEQ ID NO: 139 VHFQFPQDWWNWVRR SEQ ID NO: 140VHFQFPQDLWEFVRR SEQ ID NO: 141 VHFQFPQDWWEWFRR SEQ ID NO: 142VHFTIPADFWEWFRR SEQ ID NO: 143 VHVQIPADFWEWFRR SEQ ID NO: 144VHVTFPADLWEWFRR SEQ ID NO: 145 VHVTIPQDFWEWFRR SEQ ID NO: 146VHFTIPQDWWEWVRR SEQ ID NO: 147 VHFTFPQDLYNWVRR SEQ ID NO: 148VHFTFPQDLYNFVRR SEQ ID NO: 149 VHVTIPADLYNFFRR SEQ ID NO: 150VHFQFPQDLWEWVRR SEQ ID NO: 151 VHFTIPQDLYNWRR SEQ ID NO: 152VHFTIPADLYNFVRR SEQ ID NO: 153 VHFQIPQDLYNFFRR SEQ ID NO: 154VHFQFPQEWYNWFRR SEQ ID NO: 155 VRFQFGQEWYNFFRR SEQ ID NO: 158AHVTIPADLWDWINK SEQ ID NO: 159 GHVTIPADLWDWINK SEQ ID NO: 160LHVTIPADLWDWINK SEQ ID NO: 161 PHVTIPADLWDWINK SEQ ID NO: 162IRVTIPADLWDWINK SEQ ID NO: 163 IKVTIPADLWDWINK SEQ ID NO: 164IEVTIPADLWDWINK SEQ ID NO: 165 IQVTIPADLWDWINK SEQ ID NO: 166IYVTIPADLWDWINK SEQ ID NO: 167 IHATIPADLWDWINK SEQ ID NO: 168IHGTIPADLWDWINK SEQ ID NO: 169 IHLTIPADLWDWINK SEQ ID NO: 170IHPTIPADLWDWINK SEQ ID NO: 171 IHITIPADLWDWINK SEQ ID NO: 172IHYTIPADLWDWINK SEQ ID NO: 173 IHWTIPADLWDWINK SEQ ID NO: 174IHFTIPADLWDWINK SEQ ID NO: 175 IHVSIPADLWDWINK SEQ ID NO: 176IHVYIPADLWDWINK SEQ ID NO: 177 IHVNIPADLWDWINK SEQ ID NO: 178IHVQIPADLWDWINK SEQ ID NO: 179 IHVTAPADLWDWINK SEQ ID NO: 180IHVTGPADLWDWINK SEQ ID NO: 181 IHVTLPADLWDWINK SEQ ID NO: 182IHVTPPADLWDWINK SEQ ID NO: 183 IHVTVPADLWDWINK SEQ ID NO: 184IHVTFPADLWDWINK SEQ ID NO: 185 IHVTYPADLWDWINK SEQ ID NO: 186IHVTIAADLWDWINK SEQ ID NO: 187 IHVTIGADLWDWINK SEQ ID NO: 188IHVTILADLWDWINK SEQ ID NO: 189 IHVTIVADLWDWINK SEQ ID NO: 190IHVTIIADLWDWINK SEQ ID NO: 191 IHVTINADLWDWINK SEQ ID NO: 192IHVTIPGDLWDWINK SEQ ID NO: 193 IHVTIPLDLWDWINK SEQ ID NO: 194IHVTIPPDLWDWINK SEQ ID NO: 195 IHVTIPVDLWDWINK SEQ ID NO: 196IHVTIPIDLWDWINK SEQ ID NO: 197 IHVTIPNDLWDWINK SEQ ID NO: 198IHVTIPQDLWDWINK SEQ ID NO: 199 IHVTIPAQLWDWINK SEQ ID NO: 200IHVTIPAYLWDWINK SEQ ID NO: 201 IHVTIPADAWDWINK SEQ ID NO: 202IHVTIPADGWDWINK SEQ ID NO: 203 IHVTIPADPWDWINK SEQ ID NO: 204IHVTIPADVWDWINK SEQ ID NO: 205 IHVTIPADIWDWINK SEQ ID NO: 206IHVTIPADLHDWINK SEQ ID NO: 207 IHVTIPADLFDWINK SEQ ID NO: 208IHVTIPADLWNWINK SEQ ID NO: 209 IHVTIPADLWDHINK SEQ ID NO: 210IHVTIPADLWDFINK SEQ ID NO: 211 IHVTIPADLWDYINK SEQ ID NO: 212IHVTIPADLWDWENK SEQ ID NO: 213 IHVTIPADLWDWKNK SEQ ID NO: 214IHVTIPADLWDWANK SEQ ID NO: 215 IHVTIPADLWDWGNK SEQ ID NO: 216IHVTIPADLWDWLNK SEQ ID NO: 217 IHVTIPADLWDWPNK SEQ ID NO: 218IHVTIPADLWDWFNK SEQ ID NO: 219 IHVTIPADLWDWYNK SEQ ID NO: 220IHVTIPADLWDWIQK SEQ ID NO: 221 IHVTIPADLWDWIDK SEQ ID NO: 222IHVTIPADLWDWINH SEQ ID NO: 223 IHVTIPADLWDWIND SEQ ID NO: 224VHVTVPQELWEWVRR SEQ ID NO: 225 VHVTVPQELFEWVRR SEQ ID NO: 226VHVTVPQELYEWVRR SEQ ID NO: 227 VHVTVPQELWEWVEE SEQ ID NO: 228VHVTVPQELFEWVEE SEQ ID NO: 229 VHVTVPQELYEWVEE SEQ ID NO: 230VHVSIPQELWEWVRR SEQ ID NO: 231 VHVSVPQELWEWVRR SEQ ID NO: 232VHVSVPQELYEWVRR SEQ ID NO: 233 VRVTIPQELWEWVRR SEQ ID NO: 234VRVTVPQELYEWVRR SEQ ID NO: 235 VRVTVPQELWEWVRR SEQ ID NO: 236VHVTVPQEIYEWVRR SEQ ID NO: 237 VHVTIPQEIWEWVRR SEQ ID NO: 238VHFTVPQELYEWVRR SEQ ID NO: 239 VKISVPADLWDWINK SEQ ID NO: 240LRISVPADLWDWINK SEQ ID NO: 241 LRIYVPADLWDWINK SEQ ID NO: 242VRGYVPADLWDWINK SEQ ID NO: 243 VRAYVPADLWDWINK SEQ ID NO: 244VRLYVPADLWDWINK SEQ ID NO: 245 VRIYLPADLWDWINK SEQ ID NO: 246IHVTIPLEIFEWLQH SEQ ID NO: 247 IHVTIPLEIFEWAQH SEQ ID NO: 248IHVTIPLEIFEWLQR SEQ ID NO: 249 IHVTIPLEVFEWLQH SEQ ID NO: 250IHVTIPGEIFEWLQH SEQ ID NO: 251 VRFSVPQEIYEWVRR SEQ ID NO: 252LRISVPLEIFEWLQH SEQ ID NO: 253 LRGSVPLEIFEWLQH SEQ ID NO: 254VKISVPLEIFEWLQH SEQ ID NO: 255 VEFNFPQQVYEWFDD SEQ ID NO: 256VEFNFPQQVYEWVRR SEQ ID NO: 257 TVVYVFNEQHQEYVRK

Other peptides inhibitor of IL-17A according to the invention are thoseof Table 1A

TABLE 1A SEQ ID NO: 156 VPGAGVPGAGIHVTI SEQ ID NO: 157 VPGAGVPGAGIHVTIPA

Further aminoacid sequences may be added at the N- or C-terminal of thepeptides according to the first object of the invention.

According to a preferred embodiment, the above described peptides offormula (I) or (II) are bound at their N- and/or C-terminal to a furtheraminoacid sequence, Sequence A, having the aminoacid sequence of formula(III)Y ₁-Y ₂-Y ₃-Y ₄ −Y ₅-Y ₆-Y ₇-Y ₈-Y ₉-Y ₁₀-Y ₁₁-Y ₁₂-Y ₁₃-Y ₁₄-Y ₁₅-Y₁₆  (III)wherein:

Y₁ is A, T, V, K, R, I, L, X or G;

Y₂ is R, W, P, E, Q or A;

Y₃ is K, W, G, T, I, R or P;

Y₄ is K, T, E, W, A, R, D, G, X or F;

Y₅ is A, E, T, G, W, I, R, P or V;

Y₆ is A, W, E, R, G, P or absent;

Y₇ is K, S, W, T, F, R, V, G or absent;

Y₈ is A, Q, W, R, G or absent;

Y₉ is N, G or absent;

Y₁₀ is R or absent;

Y₁₁ is R or absent;

Y₁₂ is M or absent;

Y₁₃ is K or absent;

Y₁₄ is W or absent;

Y₁₅ is K or absent;

Y₁₆ is K or absent.

Preferably, in the sequence of formula (III), when an aminoacid isabsent, also the aminoacids at the N terminal of said aminoacids areabsent. For example, if aminoacid Y₆ is absent, also aminoacids Y₇ toY₁₆ are absent.

Preferred sequences of formula (III) according to the present inventionare listed in Table 2 below.

TABLE 2 SEQUENCE ID Sequence SEQ ID NO: 44 ARKKAAKA SEQ ID NO: 45TWWTEWSQ SEQ ID NO: 46 TWWETWW SEQ ID NO: 47 VPGWG SEQ ID NO: 48KETWWETW SEQ ID NO: 49 VPGKG SEQ ID NO: 50 VPGAG SEQ ID NO: 51RQIKIWFQNRRMKWKK SEQ ID NO: 52 RRRRRRRR SEQ ID NO: 53 VPGDGSEQ ID NO: 54 VPGXG SEQ ID NO: 55 IPGG SEQ ID NO: 56 AVGVP SEQ ID NO: 57IPGXG SEQ ID NO: 58 IPGVG SEQ ID NO: 59 LPGXG SEQ ID NO: 60 LPGVGSEQ ID NO: 61 VAPGVG SEQ ID NO: 62 XPGVG SEQ ID NO: 63 GVGVPGVGSEQ ID NO: 64 VPGFGVGAG SEQ ID NO: 65 VPGGVPGG

Accordingly, a second object of the invention is a peptide having anaminoacid sequence comprising, preferably consisting of, the sequence ofthe peptide of formula (I) or (II) according to the first object of theinvention and at the C- and/or N-terminal of said sequence a Sequence Aas above described.

The aminoacid sequence Sequence A may be bound to both the N- andC-terminal of the peptide of formula (I) or (II). In such a case, thetwo aminoacid sequences Sequence A bound to the N- and to C-terminal ofsaid peptide may be the same or different.

Alternatively, the aminoacid sequence Sequence A may be bound either tothe N- or to the C-terminal of the peptide.

Preferred individual peptides according to this object of the inventionare listed in Table 3 below.

TABLE 3 SEQ ID NO: Sequence SEQ ID NO: 21 VMVTIPADLYEWIEEARKKAAKASEQ ID NO: 22 VMVTIPADLYEWIEEGGTWWTEWSQ SEQ ID NO: 23VMVTIPADLYEWIEETWWETWW SEQ ID NO: 24 VMVTIPADLYEWIEEVPGWG SEQ ID NO: 25VMVTIPADLYEWIEEGGKETWWETW SEQ ID NO: 26 VMVTIPADLYEWIEEVPGKGSEQ ID NO: 27 VMVTIPADLYEWIEEVPGAG SEQ ID NO: 28VMVTIPADLYEWIEERQIKIWFQNRRMKWKK SEQ ID NO: 31 ARKKAAKAVMVTIPADLYEWIEESEQ ID NO: 32 GGTWWTEWSQVMVTIPADLYEWIEE SEQ ID NO: 33GGKETWWETWVMVTIPADLYEWIEE SEQ ID NO: 34 VPGWGVMVTIPADLYEWIEESEQ ID NO: 35 VPGAGVMVTIPADLYEWIEE SEQ ID NO: 36 VPGKGVMVTIPADLYEWIEESEQ ID NO: 37 VMVTIPADLYEWIEEVPGAGVPGAG SEQ ID NO: 38VPGAGVPGAGVMVTIPADLYEWIEE SEQ ID NO: 39 VPGDGVMVTIPADLYEWIEESEQ ID NO: 40 VMVTIPADLYEWIEEVPGDG

In an embodiment alternative to the above, said peptides of formula (I)or (II) according to the first object of the invention may be bound attheir N- and/or C-terminal to an aminoacid sequence (A)_(m), wherein Ais an aminoacid selected from R, K, G, E, Q or A, preferably R, K, G, Eor A, and m is an integer between 1 and 10.

The aminoacid sequence (A)_(m) may be bound to both the N- andC-terminal of the peptide of formula (I) or (II). In such a case, thetwo aminoacid sequences (A)_(m) attached to the N- and to the C-terminalof said peptide have may be the same or different.

Alternatively, the aminoacid sequence (A)_(m) may be attached either tothe N- or the C-terminal of said peptide.

Accordingly, a third object of the invention is a peptide having anaminoacid sequence that comprises, preferably consists of, the sequenceof the peptide according to the first object of the invention and at theC- and/or N-terminal of said sequence a sequence (A)_(m) as abovedefined.

Preferred individual peptides according to this embodiment of theinvention are listed in Table 4 below.

TABLE 4 SEQ ID NO: Sequence SEQ ID NO: 19 VMVTIPADLYEWIEERRRRRRSEQ ID NO: 20 VMVTIPADLYEWIEERRR SEQ ID NO: 29 RRRRRRRRVMVTIPADLYEWIEESEQ ID NO: 30 VMVTIPADLYEWIEERRRRRRRR SEQ ID NO: 43 IMVTIPADLYEWIEEQ

In a particular embodiment, said peptides of formula (I) or (II) arebound to both a sequence Sequence A and a sequence (A)_(m). In thiscase, said peptides are bound at their N- and/or C-terminal to anaminoacid sequence (A)_(m), as defined above, which is bound at its N-and/or C-terminal to an aminoacid sequence Sequence A, as describedabove.

Accordingly, a fourth object of the invention is a peptide having anaminoacid sequence that comprises, preferably consists of, the sequenceof the peptide according to the first object of the invention, at the C-and/or N-terminal of said sequence a sequence (A)_(m) as above describedand at the C- and/or N-terminal of said sequence (A)_(m) a Sequence A asabove described.

According to a preferred embodiment of the peptide according to any ofthe above objects of the invention, said peptide is bound at the C-and/or N-terminal to a protective cap group, able to preventdegradation.

Any protective cap group customary in the art may be used.

Preferably, the protective cap group bound to a C-terminal is selectedfrom amides, preferably N-alkyl amides, aldehydes, esters, preferablymethyl and ethyl esters, p-Nitroanilide, 7-Amino-4-methylcoumarin.

Preferably, the protective group cap bound to a N-terminal is selectedfrom acetyl, formyl, pyroglutamyl, fatty acids, urea, carbamatesulfonamide, alkylamine.

Preferably, the above peptides are bound to the protective cap group atthe N-terminal only. More preferably said peptides are bound to theprotective cap group at the N-terminal only and the protective cap groupis acetyl.

It is particularly advantageous to prepare dimers of the above describedpeptides according any of the different objects of the invention,preferably of the peptides of formula (I) or (II) according to the firstobject of the invention, in order to increase binding affinity andinhibitor activity.

In accordance with the above, preferably the peptides according to thedifferent objects of the invention above described, preferably of thepeptides of formula (I) or (II) according to the first object of theinvention, more preferably the peptides of formula (I) according to thefirst object of the invention, are present in form of dimers.

Accordingly, a fifth object of the present invention is a dimer formedby two peptides according to any of the above objects of the invention.Preferably said peptides are peptides of formula (I) or (II) accordingto the first object of the invention, more preferably they are peptidesof formula (I) according to the first object of the invention.

Preferably, said dimer is a homodimer, wherein the two peptides formingthe dimer have an identical sequence. Preferably, in said dimer, thepeptides are linked by means of a spacer molecule, preferably apolyethylene glycol spacer.

Preferably, according to this embodiment, polyethylene glycol is linkedto the N-terminal of the peptides according to the invention or to theaminoacids X₄, X₇ or X₁₄ of two peptides of formula (I).

According to a further embodiment, in the absence of a protective groupcap, the peptides according to the different objects of the inventionare bound, preferably at their C- and/or N-terminal, to a biomolecule toform a bioconjugate. Preferably, said biomolecule is selected fromcapric acid, capronic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronicacid, alginic acid, chitin, (GaINAc)₂, GaI-alpha1,3-GaINAc andtrigalacturonic acid.

The biomolecule is bound to the peptide either to add a specificfunction to the peptide or to modulate the physical chemical propertiesof the peptide. For example, ascorbic acid provides an antioxidantactivity to the peptide, capronic acid favors the anchoring of thepeptide to the cell membrane.

Accordingly, a sixth object of the invention is a bioconjugatecomprising, preferably consisting of, a peptide according to thedifferent objects of the invention, preferably a peptide of formula (I)or (II) according to the first object of the invention, more preferablya peptide of formula (I) according to the first object of the invention,and at least a biomolecule, as above described.

Preferably, in said bioconjugate, said biomolecule is bound to the N-and/or C-terminal of said peptide.

Preferred bioconjugates according to this embodiment are listed in Table5 below.

TABLE 5 Bioconjugate name Sequence Bio-1NAM-IMVTIPADLYEWIEE (NAM plus SEQ ID NO: 42) Bio-2IMVTIPADLYEWIEEQ-NAG (SEQ ID NO: 42 plus NAG) Bio-3NAG-NAM- IMVTIPADLYEWIEE (NAG-NAM plus SEQ ID NO: 42) Bio-4capric acid- IMVTIPADLYEWIEE (capric acid plus SEQ ID NO: 42) Bio-5Ascorbic acid IMVTIPADLYEWIEE (Ascorbic acid plus SEQ ID NO: 42)

According to one embodiment, in the above bioconjugate the abovebiomolecule or protective cap group are bound directly to the N and/orC-terminal of the peptide.

According to one embodiment, said biomolecule is bound to both the N-and C-terminal of the peptide. In such a case, the two biomoleculesbound to the N and to the C terminal of said peptide may be the same ordifferent.

According to an alternative embodiment, said biomolecule is bound toeither the N- or the C-terminal of the peptide.

According to an alternative embodiment, the above biomolecule orprotective cap group are linked to the N- and/or C terminal of thepeptide by means of a linker. Preferably, said linker is selected from4-aminobutyric acid, beta-alanine, 2-aminoethoxy-acetic acid,5-aminovaleric acid, 6-aminocaproic acid, 8-Amino-3,6-dioxaoctanoicacid, 12-amino-4,7,10-trioxadodecanoic acid,15-amino-4,7,10,13-tetraoxapenta-decanoic acid andtrioxatridecan-succinamic acid. Preferably, when the linker is attachedon one amino acid Lysine, said linker is selected from NHS-ester,isocyanates, benzoyl fluorides or carbamates.

In accordance with to the different embodiments described above, thepresent invention relates to a compound comprising or consisting of theIL-17A binding peptide of formula (I) or (II) above described, andhaving the following formula (IV):[Biomolecule or CAP]_(a)-[Linker]_(b)-(Sequence A)_(n)-A_(m)-[Peptide]-A′ _(m)′-(Sequence A)_(n′)-[Linker]_(b′)-[Biomolecule orCAP]_(a′)  (IV)wherein, independently from each other:

a=0 or 1;

b=0 or 1;

a′=0 or 1;

b′=0 or 1;

m=0 to 10;

m′=0 to 10;

n=0 or 5;

n′=0 or 5,

and

Peptide is the polypeptide according to formula (I) or formula (II);

A or A′ is one amino acid selected from R, K, G, E or A repeated m or m′times;

and Sequence A comprises the sequence of formula (III) above described;

Biomolecule is, independently from each other, capronic acid, ascorbicacid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin,(GaINAc)₂, GaI-alpha1,3-GaINAc or trigalacturonic acid;

CAP is, independently from each other, amide, aldehyde, ester,p-Nitroanilide, 7-Amino-4-Methylcoumarin, acetyl, formyl, pyroglutamylor a fatty acid;

Linker is, independently from each other, 4-aminobutyric acid,beta-alanine, 2-aminoethoxy-acetic acid, 5-aminovaleric acid,6-aminocaproic acid, 8-Amino-3,6-dioxaoctanoic acid,12-amino-4,7,10-trioxadodecanoic acid,15-amino-4,7,10,13-tetraoxapenta-decanoic acid ortrioxatridecan-succinamic acid.

According to a preferred embodiment, said Sequence A is attached to theN-terminal or to the C-terminal of the peptide of formula (I) or (II).

Some examples of the possible compounds according to the presentinvention are reported here below, but all the other possiblecombination comprised in the formula (IV) can be present:

-   -   [Biomolecule or Cap]-[Linker]-(Sequence A)-A-[Peptide]-;    -   [Biomolecule or Cap]-(Sequence A)-A-[Peptide]-;    -   [Biomolecule or Cap]-A-[Peptide]-;    -   [Biomolecule or Cap]-[Peptide]-;    -   -[Peptide]-A_(m′)-(Sequence A)_(n)-[Biomolecule or Cap]_(a′);    -   -[Peptide]-A_(m)[Biomolecule or Cap]_(a′);    -   -[Peptide]-[Biomolecule or Cap]_(a′);

According to a preferred embodiment, the biomolecule in formula (IV) ofthe present invention is selected from ascorbic acid, capronic acid, NAGor NAM.

According to a further preferred embodiment the Cap according to thepresent invention is a C-ter modification selected from amides,preferably N-alkyl amides, aldehydes, esters, preferably methyl andethyl esters, p-Nitroanilide, 7-Amino-4-Methylcoumarin or N-termodification selected from acetyl, formyl, pyroglutamyl, fatty acids,preferably capronic acid, urea, carbamate, sulfonamide or alkylamine,preferably said Cap is selected from amides, fatty acids as capronicacid and acetyl.

According to the present invention, said linker, biomolecule or Cap informula (IV) can be the same or different each other.

In a further preferred embodiment of the present invention the Linker offormula (IV) is selected from 4-aminobutyric acid, beta-alaninine,2-aminoethoxy-acetic acid, 5-aminovaleric acid ortrioxatridecan-succinamic acid.

Preferably, when the linker is attached on one amino acid Lysine, saidlinker is selected from NHS-ester, isocyanates, benzoyl fluorides orcarbamates.

A seventh object of the present invention is a pharmaceuticalcomposition comprising said peptide, dimer or a bioconjugate accordingto the objects of the invention, as described above, and at least onepharmaceutically acceptable excipient.

The pharmaceutical composition of the present invention can beformulated in a form that is suitable for topical or ophthalmicadministration.

Preferably, when the administration of the pharmaceutical composition ofthe invention is performed by topical route, the pharmaceutical form isselected from cream, ointment, gel, salve, solution, washing,suspension, drops, buffer (buffer solution), suspension, eye drops,drops, spray, wipe, or powder, preferably it is selected from cream,gel, spray, or ointment. Among the ophthalmic administration, thepharmaceutical form is preferably selected from eye drops, ophthalmicgels, ointments, wash, wipe, spray or cream.

According to a particular embodiment the peptides or bioconjugates ofthe present invention are administered locally by using microparticlesor nanoparticles.

According to the invention, the pharmaceutical composition of thepresent invention can be administered to animals and humans, defined asadults and as “paediatric population”, wherein with the term “pediatricpopulation” is indicated the part of the population from birth toeighteen years of age.

An eighth object of the present invention is the above describedpeptide, dimer or bioconjugate according to the invention for use in thetreatment and/or prevention of an inflammatory and autoimmune disease.

A ninth object of the invention is a method for the treatment and/orprevention of an inflammatory and autoimmune disease, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the above described peptide, dimer or bioconjugate accordingto the invention.

Preferably, said inflammatory and autoimmune disease is selected fromrheumatoid arthritis, multiple sclerosis, Crohn's disease, systemiclupus erythematosus, asthma, Behçet's disease, hyper IgE syndrome,ankylosing spondylitis, psoriasis, psoriatic arthritis, rheumatoidarthritis, keratoconjunctivitis sicca, vernal keratoconjunctivitis,stromal herpetic keratitis, corneal allograft rejection, cornealinfections, preferably herpes virus and Pseudomonas aeruginosakeratitis, and dry eye disease. More preferably said disease is anautoimmune ophthalmic or dermatological disease, even more preferably itis dry eye disease or psoriasis.

All of the above specified diseases and medical conditions have incommon that their origin and/or symptoms are IL-17A and/orTh-17-related.

More specifically for the latter, dry eye disease (DED), a highlyprevalent condition that includes a wide spectrum of ocular surfacedisorders, ocular mucosal inflammation is a peculiar characteristicpotentially leading to vision loss if uncontrolled, due toinflammation-induced corneal ulceration and scarring. In the progressionof DED, pathogenic immune cells, predominantly Th17 cells continuouslymigrate to the ocular mucosal surface and secrete pro-inflammatorymediators, including IL17, causing ocular surface inflammation andepitheliopathy.

The peptides, dimers or bioconjugates of the invention are convenientlyand preferably administered topically as eye drops or ophthalmic gelsand ointments.

In a further embodiment, the peptide, dimer or bioconjugate for useaccording to the present invention, are administered as the only activeprinciple or in combination with further active principles, and/or incombination with medical devices for the symptomatic treatment ofophthalmic conditions including but not limited to DED, e.g. ocularlubricants or “artificial tears”, topical re-epithelizing agents,therapeutic contacts lenses and punctum plugs.

Preferably, said further active principle is an adjuvant,immunosuppressive agent, immunomodulating agent or anti-inflammatoryagent.

For example, the IL-17A binding peptide of the present invention may beused in combination with DMSO.

According to a preferred embodiment, the IL-17A binding peptide of thepresent invention may be used in combination with immunosuppressivemonoclonal antibodies, such as monoclonal antibodies with affinity toleukocyte receptors, selected from MHC, CD2, CD3, CD4, CD7, CD8, CD25,CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; otherimmunomodulatory compounds, preferably a recombinant binding moleculehaving at least a portion of the extracellular domain of CTLA4 or amutant thereof, an at least extracellular portion of CTLA4 or a mutantthereof joined to a non-CTLA4 protein sequence, e.g. (e.g. CTLA4Ig,designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesionmolecule inhibitors, LFA-I antagonists, ICAM-I or -3 antagonists, VCAM-4antagonists or VLA-4 antagonists.

In a further preferred embodiment the IL-17A binding peptide of thepresent invention is used in combination with DMARD (disease-modifyingantirheumatic drug), preferably Gold salts, sulphasalazine,anti-malarias, methotrexate, D-penicillamine, azathioprine, mycophenolicacid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide,glucocorticoids; a calcineurin inhibitor, preferably cyclosporin A or FK506; a modulator of lymphocyte recirculation, preferably FTY720 andFTY720 analogs; a mTOR inhibitor, preferably rapamycin,40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; anascomycin having immuno-suppressive properties, preferably ABT-281,ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualine or an immunosuppressive homologue, analogue orderivative thereof; or a chemotherapeutic agent, preferably pacli-taxel,gemcitabine, cisplatinum, doxorubicin or 5-fluorouracil; anti-TNFagents, preferably monoclonal antibodies to TNF, preferably infliximab,adaiimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII,preferably Etanercept, PEG-TNF-RI; blockers of proinflammatorycytokines, IL-1 blockers, preferably Anakinra or IL-1 trap, AAL160, ACZ885, IL-6 blockers; inhibitors or activators of proteases, preferablymetalloproteases, anti-IL-15 antibodies, anti-IL-6 antibodies,anti-IL-23 antibodies, anti-IL-22 antibodies, anti-IL-21 antibodies,anti-IL-12 antibodies, anti-IFN-gamma antibodies, anti-IFN-alphaantibodies, anti-CD20 antibodies, anti-inflammatory agents, preferablyaspirin or an anti-infectious agent. Naturally, this list of agents forco-administration is not limiting nor complete.

A further object of the present invention is one the peptides listed inTable 6 for use in the treatment and/or prevention of an inflammatoryand autoimmune disease described above.

TABLE 6 SEQ ID NO: SEQUENCE SEQ ID NO: 1 IHVTIPADLWDWINK SEQ ID NO: 66IVVTMPADLWDWIKA SEQ ID NO: 67 IVVTMPADLWDWIRA SEQ ID NO: 68IVVTMPADLWDWIRK SEQ ID NO: 69 IVVTMPADLWDWIAA SEQ ID NO: 70IVVTMPADLWDWARA SEQ ID NO: 71 IVVTMPADLWAWIRA SEQ ID NO: 72IVVTMPADLADWIRA SEQ ID NO: 73 IVVTMPADAWDWIRA SEQ ID NO: 74IVVTMPADLWDWIRA SEQ ID NO: 75 IVVTAPADLWDWIRA SEQ ID NO: 76IVVAMPADLWDWIRA SEQ ID NO: 77 IAVTMPADLWDWIRA SEQ ID NO: 78AVVTMPADLWDWIRA SEQ ID NO: 79 IHVTMPADLWDWIRA SEQ ID NO: 80IQVTMPADLWDWIRA SEQ ID NO: 81 IRVTMPADLWDWIRA SEQ ID NO: 82ITVTMPADLWDWIRA SEQ ID NO: 83 IWVTMPADLWDWIRA SEQ ID NO: 84IYVTMPADLWDWIRA SEQ ID NO: 85 IVVTIPADLWDWIRA SEQ ID NO: 86IVVTLPADLWDWIRA SEQ ID NO: 87 IVVTVPADLWDWIRA SEQ ID NO: 88IVVTMPADLWDWIMA SEQ ID NO: 89 IVVTMPADLWDWINA SEQ ID NO: 90IVVTMPADLWDWIQA SEQ ID NO: 91 IVVTIPADLWDWIRA SEQ ID NO: 92IVVTLPADLWDWIRA SEQ ID NO: 93 IHVTIPADLWDWINK SEQ ID NO: 94IHVTIPADLWDWIN SEQ ID NO: 95 IHVTIPADLWDWI SEQ ID NO: 96DSSAVCWAFPHHPLCHMKAT SEQ ID NO: 97 ADADMCWFFPTSPWCH SEQ ID NO: 98DLSAVCWAFPWDPECHM SEQ ID NO: 99 DSSAVCWAFPYLPECH SEQ ID NO: 100DISAVCWAFPFDPECH SEQ ID NO: 101 AYECPRLEYDMFGALHCLPS SEQ ID NO: 102CPRLEYDMFGALHCL SEQ ID NO: 103 CLDLQYDPWGALHCI SEQ ID NO: 104CFDLQYDPWGALHCI SEQ ID NO: 105 CLDLQYDMFGALHCV SEQ ID NO: 106CLDLVYDPWGALHCI SEQ ID NO: 107 CWVLEYDMFGALHCR SEQ ID NO: 108CWALEYDMFGYLHCR SEQ ID NO: 109 CWVLEYDMFGFLHCR SEQ ID NO: 110CWVLEYDMFGYLHCR

The amount and mode of administration of the peptides according to thepresent invention will vary depending upon the particular peptideinhibitor of the invention, the individual patient group or patient, thepresence of further medically active compounds and the nature andseverity of the condition being treated.

According to a preferred embodiment, the prophylactic and/or therapeuticuse dosages is of about 5-50 μg/ml, preferably of about 10-25 μg/mlPreferably, the frequency of administration for prophylactic and/ortherapeutic uses lies in the range of about once to twice dailyapplications, preferably once.

The invention is further described by way of illustration in thefollowing examples, none of which are to be interpreted as limiting thescope of the invention as outlined in the appended claims.

EXAMPLES Example 1 Peptides Synthesis & Purification

We have developed a homology model of IL-17A by use of crystallographicdata available (PDB 5h13, PDB 5vb9 and PDB 4hsa) (Liu et al. Nat Commun2013, 4, 1888, Liu et al. Sci Rep 2016, 6: 30859, Liu et al. Sci Rep2016, 6, 26071, Ting, Tung et al. Plos One 2018, 13(1): e01908502018).In our models we have also recreated the loops unresolved in thecrystals and optimized models through a molecular dynamics simulationperformed with Desmond software, as implemented in Schrodinger Maestromacromodel suite. All molecular simulations were run for 1 microsecondto guarantee the system stability.

Based on structure and properties of IL-17A α-pocket, different peptidesequences have been designed so as to obtain i) a stable secondary andtertiary conformation that engages IL-17A with an optimized proteinbinding site occupancy, and ii) physical-chemical properties suitablefor topical use in ophthalmic and dermatological pathologies (Liu et al.Nat Commun 2013, 4, 1888, Espada et al J Med Chem 2016, 59(5),2255-2260).

Additionally, we optimized the interactions ligand-IL-17A by theintroduction of hydrogen bonds and optimization of hydrophobicinteractions.

a. Fmoc-Based Solid-Phase Peptide Synthesis

All the chemicals were purchased and used without further purification.

Peptides of SEQ ID NO: 1 to 43 and 111-257 of Table 1 were prepared bymanual or automatic solid-phase peptide synthesis, on Rink Amide-MBNAresin as the solid support, as described below and with N-terminal aminecapped with acetyl group.

The Fmoc (9-fluorenylmethoxycarbonyl) group used for Na-protection wascleaved by 8 min treatment with 20% piperidine in dimethylformamide(DMF) followed by a further treatment with the same reagent for 10 min.After Fmoc cleavage, the peptide-resin complex was washed with DMF (×6).The next residue was then incorporated through the use of1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxidhexafluorophosphate (HATU)/N,N-diisopropylamine (DIPEA) couplingprotocol [Fmoc-amino acid (3 equiv), HATU (3 equiv), and DIPEA (6equiv)]. After gentle stirring (1 hr) and washing with DMF (×6), part ofthe peptide-resin was analyzed by Kaiser test. On completion of theassembly, the peptide-resin was washed with DMF (×3), dichloromethane(DCM) (×4) and then dried in vacuo.

Cleavage and Deprotection

The peptide was cleaved from the resin and, when necessary, deprotectedby using as cleavage solution a mixture of trifluoroaceticacid/1,2-ethanedithiol/thioanisole/phenol/H2O/triisopropylsilane in theratio 68.5/10/10/5/3.5/1, v/v. 3 ml of cleavage solution was used per100 mg of resin. Complete deprotection was achieved under stirring for 4hours at 30° C. Following the cleavage reaction, the peptide in themixture was precipitated in cold diethyl ether and dried in vacuo at 50°C.

b. Disulfide Bond Formation in Peptide 41

Disulfide bond formation in peptide of SEQ ID NO: 41 of Table 1 wasobtained by incubation of the peptide in a mixture DMSO/H2O (20/80,v/v), which was monitored by RP-HPLC, MS and free sulfhydryl detection(DTNB method).

c. Purification by Preparative RP-HPLC and Purity Assessment byAnalytical HPLC

The crude peptide samples were purified by preparative RP-HPLC (Agilent)using a C18 column (10 μm, 100 Å, 50×250 mm). A solvent systemconsisting of solvent A (0.1% TFA, 2% CH3CN in water) and solvent B (90%CH3CN/H2O) at a flow rate of 25 mL/min was used for elution, and theabsorbance was detected at 220 nm. The solvent was removed bylyophilization. The final products were characterized by MALDI-TOF-MS,and the purity of the material assessed by analytical RP-HPLC (C18-250mm×4.6 mm I.D., flow rate of 1 mL/min), and the absorbance was detectedat 220 nm.

Example 2: Preparation of NAM and/or NAG Bioconjugated Peptides BIO-1,BIO-2 and BIO-3

The bioconjugated peptides BIO-1, BIO-2 and BIO-3 were prepared by aconjugation reaction of peptide 42 or 43 according the followingprocedures.

1. Preparation of Donor and Acceptor

All reactions were performed under N₂ atmosphere. NMR spectra were runon a Brucker 400 MHz instrument. HPLC-UV analysis were obtained on anAgilent 1260 Infinity System, equipped with a G1311B Agilent 1260quaternary pump, G1329B Agilent 1260 autosampler, G1315C Agilent 1260diode array detector, and G1316A Agilent 1260 column thermostat modules.A Phenomenex GEMINI C18 150×4.6 mm2 (5 μm) column was used. The mobilephase A was MilliQ water with 0.05% TFA, and B was HPLC gradeacetonitrile with 0.05% TFA at a flow rate of 1.0 mL/min. The HPLCsystem was coupled to an Agilent Quadrupole 6120 LC/MS mass spectrometeroperated in positive ion mode. Ions were generated using an electrosprayionization ion source. Acquisition data were processed with AgilentChemstation Software.

a. Synthesis of1-(2,2,2-Trichloroacetimine)2-Deoxy-3-O-Acetyl-4,6-Obenzylidene-2-(2,2,2-Tri-Chloroethoxycarbonylamino)-a-D-Glucopyranoside(Donor)

Trichloroethoxycarbonyl chloride (2.1 mL, 15.3 mmol) was added dropwiseat room temperature to a vigorously stirred solution of D-glucosaminehydrochloride (3 g, 13.9 mmol) and NaHCO₃, (3.5 g, 41.7 mmol) in water(30 mL). The mixture was stirred for 2 h, then neutralized with 1 M HCl,concentrated, and dried in vacuo.2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside(intermediate A) was obtained.

The crude product obtained was directly used for the next reaction stepwithout further purification.

In details, zinc chloride (1.9 g, 13.9 mmol) was added to a solution ofintermediate A (4.9 g, 13.9 mmol) in benzaldehyde (24 ml) and molecularsieves, 4 Å (4 Å MS, 600 mg). After stirring overnight at roomtemperature, saturated aq NaHCO₃ (30 mL) and diethyl ether (90 mL) wereadded and the reaction mixture was stirred for 15 min. The formedprecipitate was filtered, washed with water, diethyl ether, and dried.The residue was dissolved in pyridine (13 mL) cooled to 0° C., treatedwith acetic anhydride (6.4 mL, 68 mmol) and stirred overnight at roomtemperature, then the solution was concentrated with toluene, cooled to0° C. and extracted with DCM and saturated aq NaHCO₃ (3×), then washedwith a saturated NaCl solution. The combined organic layers were dried,concentrated, and the crude purified by Isolera (EtPet/EtOAc) to give1,3-di-O-acetyl2-deoxy-4,6-O-benzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside(intermediate B), as a white solid (3.05 g, 41.7% yield).

Morpholine (1.2 mL, 13.9 mmol) was then added to a solution ofintermediate B (3.05 g, 5.8 mmol) in dry ethyl acetate (EtOAc) (12.2mL). After stirring overnight at room temperature the reaction mixturewas quenched with 3N HCl solution (3.5 mL) and then stirred for 20 min.

Extracted with EtOAc and washed with water, saturated aq NaHCO₃ and asaturated NaCl solution saturated NaCl solution, dried, andconcentrated. The crude was purified by Isolera (EtPet/EtOAc) to give2-deoxy-3-O-acetyl-4,6-O-benzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside(intermediate C) as a white solid (2.2 g, 78.4%). To a solution ofintermediate C (2.2 g, 4.54 mmol) in dry DCM (44 ml), Cs₂CO₃ (680 mg,2.1 mmol) and CCl₃CN (4.4 mL, 43.8 mmol) were added. The reactionmixture was stirred for 2 h at room temperature, then filtered overcelite and concentrated to give1-(2,2,2-Trichloroacetimine)2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside(Donor) as a light yellow solid (2.2 g, 77%).

b. Synthesis of 1,6-Di-O-benzyl2-deoxy-3-O-((R)-10-ethoxycarbonylethyl)-2-(2,2,2-trichloroetho-xycarbonylamino)-a-D-glucopyranoside(Acceptor)

Allyl chloroformate (6.6 mL, 62.2 mmol) was added dropwise at roomtemperature to a vigorously stirred solution of D-glucosaminehydrochloride (12.2 g, 56.6 mmol) and NaHCO₃, (14.3 g, 169.7 mmol) inwater (61 mL). The mixture was stirred for 2 h, then neutralized with 1MHCl, concentrated, and dried in vacuo. Crude2-deoxy-2-(allyloxycarbonylamino)-a-D-glucopyranoside (Intermediate D)was obtained. The product was used for next reaction step withoutfurther purification, Intermediate D (14.9 g, 56.6 mmol) was dissolvedin benzyl alcohol (89 mL, and acetyl chloride (15.4 mL, 215.2 mmol) wasadded dropwise at 0° C. After stirring for 3 h at 80° C. the reactionmixture was quenched with cold saturated aq NaHCO³ (20 ml) and stirredfor additional 30 min. Cold water and diethyl ether were added and thereaction mixture was stirred for 30 min. The two phases were separated,and the aqueous phase was concentrated, and dried in vacuo. 30 ml ofwater and 200 ml of ethyl ether were added until the formation of aprecipitate that was filtered and washed several times with cold diethylether (until no traces of benzyl alcohol were detected. Benzyl2-deoxy-2-(allyloxycarbonylamino)-a-D-glucopyranoside (intermediate E)was obtained as a white solid (11 g, 55%) and directly used for nextreaction step without further purification. Zinc chloride (4.2 g, 31.1mmol) was added to a solution of intermediate E (11 g, 31.1 mmol) inbenzaldehyde (55 mL) and 4 Å MS (4.9 g). After 2 h, more zinc chloride(4.2 g, 31.1 mmol) was added. After stirring overnight at roomtemperature, the reaction mixture was treated with saturated aq NaHCO₃(70 mL), petroleum ether (420 mL), and stirred for 10 min. Theprecipitate was filtered, washed with petroleum ether and dissolved inDCM. The organic solution was extracted with saturated aq NaHCO₃, water,and a saturated NaCl solution dried and concentrated. The crude waspurified by Isolera (DCM/EtOAc) to give Benzyl2-deoxy-4,6-O-benzylidene-2-(allyloxycarbonylamino)-a-D-glucopyranoside(intermediate F) as a white solid (2.75 g, 20%).

To a solution of intermediate F (2.75 g, 6.2 mmol) in dry DCM (40.6 mL),cooled to 0° C., sodium hydride (NaH, 348.8 mg, 8.7 mmol, 60% oildispersion) was added, and the mixture stirred for 30 min at roomtemperature. The mixture was then treated with neat (−)-ethyl(S)-2-trifluromethanesulfonyl propionate dropwise and stirred for 2 h atroom temperature. The reaction mixture was quenched by addition of ice,and extracted with DCM. The organic solution was washed with saturatedaq NaHCO₃, a saturated NaCl solution, dried and concentrated. Theresidue was purified by Isolera (DCM/EtOAc) to give benzyl2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-4,6-Obenzylidene-2-(allyloxy-carbonylamino)-a-D-glucopyranoside (intermediate G) asa white solid (2.35 g, 69.7%).

Tetrakis(triphenylphosphine) palladium (1.5 g, 1.3 mmol) and acetic acid(AcOH, 0.385 mL, 6,725 mmol) were added to a solution of theintermediate G (2.35 g, 4.34 mmol) in dry DCM (42.7 mL). The reactionmixture was stirred at room temperature for 15 min and then2,2,2-trichloroethyl chloroformate (TrocCl, 1.224 mL, 8,895 mmol) wasadded dropwise, and the resulting solution stirred for 1 h at roomtemperature. The reaction mixture was quenched with saturated aq NaHCO₃,extracted with DCM, washed with H₂O and a saturated NaCl solution. Afterconcentration in vacuo, the residue was dissolved in diethyl ether (100ml) and insoluble materials filtered off. The organic phase was dried,concentrated, and purified by Isolera (Cyclohexane/AcOEt) to give benzyl2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-4,6-O-benzylidene-2-(2,2,2-tri-chloroethoxycarbonylamino)-a-D-glucopyranoside(intermediate H) as a white solid (2.3 g, 83.7%).

To a solution of intermediate H (2.4 g, 3.8 mmol) in dry CH3CN (38 mL)at 0° C. a solution of Me3N—BH₃ (332 mg, 4.55 mmol) in CH₃CN (2.2 mL)was added, followed by a solution of BF₃-OEt₂ (2.89 mL, 23.4 mmol) inCH3CN (8 mL) added dropwise. After stirring for 3 h at 0° C., themixture was quenched with cold saturated aq NaHCO₃ (30 mL), diluted withEtOAc (350 ml), and washed with saturated aq NaHCO₃ (100 ml), 5% citricacid (4×50 ml), saturated aq NaHCO₃ (50 mL), and a saturated NaClsolution (40 mL). The organic layer was dried, concentrated, and thecrude was purified by Isolera (Cyclohexane/AcOEt) to give1,6-di-O-benzyl2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-2-(2,2,2-trichloroetho-xycarbonylamino)-a-D-glucopyranoside(Acceptor), as a colorless foam (1.2 g, 49.8%).

2. Synthesis of NAG-NAM Conjugated Peptide BIO-3

Donor (1.6 g, 2.6 mmol) and Acceptor (1.1 g, 1.73 mmol), suspended indry DCM (55 mL) with 4 Å molecular sieves (200 mg), were treated withTrimethylsilyl trifluoromethanesulfonate (TMSOTf, 188 μL, 1.04 mmol) at−15° C. After stirring for 20 min, more 0.75 eq of Donor and 0.3 eq ofTMSOTf were added. After stirring for 20 min, more 0.75 eq of Donor and0.3 eq of TMSOTf were added. The mixture was then quenched with coldsaturated aq solution of NaHCO₃ (15 mL) and extracted with DCM (60 mL).The organic layer was washed with saturated aq NaHCO₃ and a saturatedNaCl solution, dried and concentrated. The residue was purified byIsolera reverse phase (H2O/ACN neutral phases) to give benzyl6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-1′-ethoxycarbonylethyl]-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside(intermediate L), as a white foam (1.2 g, 62.9%).

A solution of intermediate L (1.2 g, 1.09 mmol) and zinc-copper couple(3.12 g, 24.2 mmol) in a mixture AcOH/Ac2O/THF 1:1:1 (12 mL) was stirredfor 4 h at room temperature. The reaction mixture was filtered overcelite, washed with EtOAc, and concentrated. The crude was concentratedand purified by column chromatography (Cyclohexane/AcOEt) to give benzyl6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-acetylamino-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-10-ethoxycarbonylethyl]-2-acetylamino-a-D-glucopyranoside(intermediate M) as a white solid (560 mg, 0.67 mmol, 61.6%). HPLC-MS(ESI+) m/z: C₄₄H₅₄N₂O₁₄+Na: 857.3472. Found: 857.6124.

To a solution of intermediate M (91 mg, 0.109 mmol) inTHF/1,4-dioxane/H₂O 4:2:1 (2.8 mL) was added LiOH.H₂O (56 mg, 1.34mmol). After stirring for 2 h at room temperature, the reaction mixturewas filtered over Dowex H+ (freshly activated with 1N HCl). The residuewas purified by diaion HP-20 column chromatography (2×7 cm) previouslywashed with water, MeOH and water. Column was eluted first with H₂O (50mL) and then with MeOH (30 mL). The alcohol fractions were concentratedto give benzyl6-O-benzyl-4-O-[2-deoxy-4,6-O-benzylidene-2-acetylamino-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-10-ethoxycarbonylethyl]-2-acetylamino-a-D-glucopyranoside(intermediate N), as a whitish solid (57.4 mg, 69%). Mp 102-105° C.,1HNMR (600 MHz, CD₃OD) δH 7.51-7.27 (m, 15H, ArH), 5.59 (s, 1H, CHPh),5.36 (d, J 3.1 Hz, H−1), 4.85 (m) H₂O, H−10, ½ CH₂Ph), 4.66-4.57 (m, 3H,J 12.1 Hz, CHCH₃, CH₂-Ph), 4.46 (d, 1H, J 12.2 Hz, ½ CH₂Ph), 4.29 (dd,1H, J 10.3 Hz, J 5.0 Hz, H-60b), 4.07 (t, 1H, J 9.6 Hz, H−30), 3.95 (t,1H, J 9.1 Hz, H−4), 3.82-3.77 (m, 2H, H−6b, H−3), 3.72-3.62 (m, 3H,H−60a, H−5, H−6a), 3.55-3.40 (m, 3H, H−20, H−40, H−2), 3.30-3.27 (m, 1H,H−50), 1.98 (s, 3H, COCH₃), 1.96 (s, 3H, COCH₃), 1.37 (d, J 6.9 Hz, 3H,CHCH₃). HPLC-MS (ESI+) m/z: C₄₀H₄₈N₂O₁₃+Na: 787.3054 Found: 787.7830.

The peptide of SEQ ID NO: 42 was coupled to compound N in the conditionsused for aminoacid coupling and the resulting bioconjugate was cleavedfrom the solid support by treatment with trifluoroacetic acid (1%solution in DCM). The resulting bioconjugate (0.026 mmol) was dissolvedin acetic acid (8 mL), and Pd(OH)2/C (58 mg) was added. The resultingmixture was stirred at room temperature for 6 h under hydrogenatmosphere. The mixture was filtered over celite and concentrated togive the bioconjugate B10-3 in quantitative yield. The bioconjugate wascleaved from the resin and the last residue deprotected by using ascleavage solution a mixture of trifluoroaceticacid/1,2-ethanedithiol/thioanisole/phenol/H2O/triisopropylsilane in aratio of 68.5/10/10/5/3.5/1, v/v. 3 ml of cleavage solution was used per100 mg of resin. Complete deprotection was achieved after 4 hours at 30°C. Following the cleavage reaction, the crude peptide was precipitatedby addition of cold diethyl ether and dried in vacuo at 50° C.

The crude bioconjugate was purified by preparative RP-HPLC (Agilent)using a C18 column (10 μm, 100 Å, 50×250 mm). A solvent mixtureconsisting of solvent A (0.1% TFA, 2 CH₃CN in H₂O) and solvent B (90%CH₃CN/H₂O) at a flow rate of 25 mL/min was used for elution, and theabsorbance detected at 220 nm. The solvent was removed by lyophilizationand the final products characterized by MALDI-TOF-MS. The purity of thepurified material was assessed by analytical RP-HPLC (C18-250 mm×4.6 mmI.D., flow rate of 1 mL/min), and the absorbance was detected at 220 nm.

3. Synthesis of NAG and NAM Bioconjugates B10-1 and BIO-2

Synthesis of NAM-Peptide and Peptide-NAG were performed starting fromthe peptide anchored resin (Example 1), wherein the peptide was thepeptide of SEQ ID NO: 42 (for BIO-1) or the peptide of SEQ ID NO: 43(for BIO-2), respectively, and Donor and Acceptor were the moleculesdescribed above according the procedure described in Swaminathan et al.,Proc Natl Acad Sci USA. 2006, 17; 103 (3):684-9.

Example 3: Synthesis of Capric Acid Bioconjugate BIO-4

Peptides capped with Capric Acid were synthetized according theprocedure described in Example 1 by using a N-terminal amine capped withCapric Acid.

Example 4: Synthesis of Ascorbic Acid Bioconjugate BIO-5

5,6-O-isopropylidene-L-ascorbic acid (iASA) was synthesized by Jung'smethod (Jung, M. E.; Shaw, T. J. J. Am. Chem. Soc. 1980, 102, 6304). Forthe activation, CDI (10 eq.) was added to the peptide anchored resin for2 h. Activation of N-terminal amine of peptide, after cleavage from theresin, was confirmed by RP-HPLC using the following conditions: A to B(A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; from 0% to 60% B over 30 min,at a flow rate of 1.0 mL/min); detection, UV 230 nm. Next, iASA wasintroduced to the activated peptide anchored resin. The product wasseparated from the resin by treating with 50% TFA/DCM (v/v) for 1 h. Theresin was filtered, the filtrate was concentrated in high vacuum, andprecipitated with cold diethyl ether. The structure of the resultingbioconjugate BIO-5 was confirmed by LC/MS using 0.1% formicacid/methanol as eluent at a flow rate of 250 μL/min over 30 min, andmonitored at 230 nm.

Example 5: IL-17RA/IL17RC Dimerization Assay in Response to IL-17A

The peptides of Table 7 were tested for their ability to inhibit thebinding of IL-17A to its receptor and the subsequent interaction ofIL-17RA and IL17RC.

Interleukin receptor RA and RC dimerization assay (DiscoverX) was usedto measure the interaction of the receptor chains IL-17RA and IL17RCupon activation by IL-17A in the absence or presence of the peptides ofTable 7.

In the PathHunter® Cytokine Receptor Assay, one cytokine receptor chainis tagged with a small peptide epitope ProLink (PK) and the other chainis tagged with Enzyme Acceptor (EA). Ligand binding induces dimerizationof the two receptors, facilitating complementation of PK and EAfragments. This interaction generates an active unit of b-galactosidase,which is detected using a chemiluminescent substrate. PathHunter celllines were expanded from freezer stocks according to standardprocedures. Cells were seeded in a total volume of 20 μL into whitewalled, 384-well microplates and incubated for the appropriate timeprior to testing. For agonist activity inhibition, cells were incubatedwith sample to induce response. Intermediate dilution of sample stockswas performed to generate 5× sample in assay buffer. 5 μL of 5× samplewas added to cells and incubated at 37° C. or room temperature for 60minutes. Vehicle concentration was 1%. 5 μL of 6×EC₈₀ agonist (IL-17A)in assay buffer was added to the cells and incubated at 37° C. for 6 to16 hours depending on the assay. 1. Assay signal was generated through asingle addition of 12.5 or 15 μL (50% v/v) of PathHunter Detectionreagent cocktail for agonist (IL-17A) and (peptides of table 7) assaysrespectively, followed by a one hour incubation at room temperature. Forsome assays, activity was detected using a high sensitivity detectionreagent (PathHunter Flash Kit) to improve assay performance. In theseassays, an equal volume of detection reagent (25 or 30 uL) was added tothe wells, followed by a one hour incubation at room temperature.Microplates were read following signal generation with a PerkinElmerEnvision™ instrument for chemiluminescent signal detection. Compoundactivity was analyzed using CBIS data analysis suite (ChemInnovation,CA). % Inhibition=100%×(1−(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of EC80 control−mean RLU of vehicle control)).

As reported in Table 7 below, all tested molecules were found active ininhibiting IL-17RA/IL17RC dimerization thus inhibiting the IL-17Apathway activation showing a IC50 value in the mid nanomolar range.

TABLE 7 IC₅₀ in IL-17RA/IL7RC dimerization SEQUENCE NAME assay SEQ IDNO: 1 <0.2 μM SEQ ID NO: 2 <0.2 μM SEQ ID NO: 3 <0.4 μM SEQ ID NO: 4<0.1 μM SEQ ID NO: 5 <0.1 μM SEQ ID NO: 6 <0.4 μM SEQ ID NO: 7 <0.1 μMSEQ ID NO: 8 <0.2 μM SEQ ID NO: 9 <0.3 μM SEQ ID NO: 10 <0.4 μM SEQ IDNO: 11 <0.4 μM SEQ ID NO: 12 <0.2 μM SEQ ID NO: 13 <0.4 μM SEQ ID NO: 14<0.4 μM SEQ ID NO: 15 <0.4 μM SEQ ID NO: 16 <0.4 μM SEQ ID NO: 17 <0.4μM SEQ ID NO: 18 <0.4 μM SEQ ID NO: 19 <1 μM SEQ ID NO: 20 <1 μM SEQ IDNO: 21 <1 μM SEQ ID NO: 22 <1 μM SEQ ID NO: 23 <1 μM SEQ ID NO: 24 <1 μMSEQ ID NO: 25 <1 μM SEQ ID NO: 26 <1 μM SEQ ID NO: 27 <1 μM SEQ ID NO:28 <1 μM SEQ ID NO: 29 <1 μM SEQ ID NO: 30 <1 μM SEQ ID NO: 31 <1 μM SEQID NO: 32 <1 μM SEQ ID NO: 33 <1 μM SEQ ID NO: 34 <1 μM SEQ ID NO: 35 <1μM SEQ ID NO: 36 <1 μM SEQ ID NO: 37 <0.2 μM SEQ ID NO: 38 <0.2 μM SEQID NO: 39 <0.2 μM SEQ ID NO: 40 <0.2 μM SEQ ID NO: 41 <1 μM SEQ ID NO:111 <0.5 μM SEQ ID NO: 112 <0.5 μM SEQ ID NO: 113 <0.5 μM SEQ ID NO: 114<0.5 μM SEQ ID NO: 115 <0.5 μM SEQ ID NO: 116 <0.5 μM SEQ ID NO: 117<0.5 μM SEQ ID NO: 118 <0.5 μM SEQ ID NO: 119 <0.5 μM SEQ ID NO: 120<0.5 μM SEQ ID NO: 121 <0.5 μM SEQ ID NO: 122 <0.5 μM SEQ ID NO: 123<0.5 μM SEQ ID NO: 124 <0.5 μM SEQ ID NO: 125 <0.5 μM SEQ ID NO: 126<0.5 μM SEQ ID NO: 127 <0.5 μM SEQ ID NO: 128 <0.5 μM SEQ ID NO: 129<0.5 μM SEQ ID NO: 130 <0.5 μM SEQ ID NO: 131 <0.5 μM SEQ ID NO: 132<0.5 μM SEQ ID NO: 133 <0.5 μM SEQ ID NO: 134 <0.5 μM SEQ ID NO: 135<0.5 μM SEQ ID NO: 136 <0.5 μM SEQ ID NO: 137 <0.5 μM SEQ ID NO: 138<0.5 μM SEQ ID NO: 139 <0.5 μM SEQ ID NO: 140 <0.5 μM SEQ ID NO: 141<0.5 μM SEQ ID NO: 142 <0.5 μM SEQ ID NO: 143 <0.5 μM SEQ ID NO: 144<0.5 μM SEQ ID NO: 145 <0.5 μM SEQ ID NO: 146 <0.5 μM SEQ ID NO: 147<0.5 μM SEQ ID NO: 148 <0.5 μM SEQ ID NO: 149 <0.5 μM SEQ ID NO: 150<0.5 μM SEQ ID NO: 151 <0.5 μM SEQ ID NO: 152 <0.5 μM SEQ ID NO: 153<0.5 μM SEQ ID NO: 154 <0.5 μM SEQ ID NO: 155 <0.5 μM SEQ ID NO: 156<0.5 μM SEQ ID NO: 157 <0.5 μM NAM-IMVTIPADLYEWIEE BIO-1 <1 μM (NAM plusSEQ ID NO: 42) IMVTIPADLYEWIEEQ-NAG BIO-2 <1 μM (SEQ ID NO: 42 plus NAG)NAG-NAM-IMVTIPADLYEWIEE BIO-3 <1 μM (NAG-NAM plus SEQ ID NO: 42) capricacid-IMVTIPADLYEWIEE BIO-4 <1 μM (capric acid plus SEQ ID NO: 42)Ascorbic acid-IMVTIPADLYEWIEE BIO-5 <1 μM (Ascorbic acid plus SEQ ID NO:42) SEQ ID NO: 158 <10 μM SEQ ID NO: 159 <10 μM SEQ ID NO: 160 <10 μMSEQ ID NO: 161 <10 μM SEQ ID NO: 162 <10 μM SEQ ID NO: 163 <10 μM SEQ IDNO: 164 <10 μM SEQ ID NO: 165 <10 μM SEQ ID NO: 166 <10 μM SEQ ID NO:167 <10 μM SEQ ID NO: 168 <10 μM SEQ ID NO: 169 <10 μM SEQ ID NO: 170<10 μM SEQ ID NO: 171 <10 μM SEQ ID NO: 172 <10 μM SEQ ID NO: 173 <10 μMSEQ ID NO: 174 <10 μM SEQ ID NO: 175 <10 μM SEQ ID NO: 176 <10 μM SEQ IDNO: 177 <10 μM SEQ ID NO: 178 <10 μM SEQ ID NO: 179 <10 μM SEQ ID NO:180 <10 μM SEQ ID NO: 181 <10 μM SEQ ID NO: 182 <10 μM SEQ ID NO: 183<10 μM SEQ ID NO: 184 <10 μM SEQ ID NO: 185 <10 μM SEQ ID NO: 186 <10 μMSEQ ID NO: 187 <10 μM SEQ ID NO: 188 <10 μM SEQ ID NO: 189 <10 μM SEQ IDNO: 190 <10 μM SEQ ID NO: 191 <10 μM SEQ ID NO: 192 <10 μM SEQ ID NO:193 <10 μM SEQ ID NO: 194 <10 μM SEQ ID NO: 195 <10 μM SEQ ID NO: 196<10 μM SEQ ID NO: 197 <10 μM SEQ ID NO: 198 <10 μM SEQ ID NO: 199 <10 μMSEQ ID NO: 200 <10 μM SEQ ID NO: 201 <10 μM SEQ ID NO: 202 <10 μM SEQ IDNO: 203 <10 μM SEQ ID NO: 204 <10 μM SEQ ID NO: 205 <10 μM SEQ ID NO:206 <10 μM SEQ ID NO: 207 <10 μM SEQ ID NO: 208 <10 μM SEQ ID NO: 209<10 μM SEQ ID NO: 210 <10 μM SEQ ID NO: 211 <10 μM SEQ ID NO: 212 <10 μMSEQ ID NO: 213 <10 μM SEQ ID NO: 214 <10 μM SEQ ID NO: 215 <10 μM SEQ IDNO: 216 <10 μM SEQ ID NO: 217 <10 μM SEQ ID NO: 218 <10 μM SEQ ID NO:219 <10 μM SEQ ID NO: 220 <10 μM SEQ ID NO: 221 <10 μM SEQ ID NO: 222<10 μM SEQ ID NO: 223 <10 μM SEQ ID NO: 224 <10 μM SEQ ID NO: 225 <10 μMSEQ ID NO: 226 <10 μM SEQ ID NO: 227 <10 μM SEQ ID NO: 228 <10 μM SEQ IDNO: 229 <10 μM SEQ ID NO: 230 <10 μM SEQ ID NO: 231 <10 μM SEQ ID NO:232 <10 μM SEQ ID NO: 233 <10 μM SEQ ID NO: 234 <10 μM SEQ ID NO: 235<10 μM SEQ ID NO: 236 <10 μM SEQ ID NO: 237 <10 μM SEQ ID NO: 238 <10 μMSEQ ID NO: 239 <10 μM SEQ ID NO: 240 <10 μM SEQ ID NO: 241 <10 μM SEQ IDNO: 242 <10 μM SEQ ID NO: 243 <10 μM SEQ ID NO: 244 <10 μM SEQ ID NO:245 <10 μM SEQ ID NO: 246 <10 μM SEQ ID NO: 247 <10 μM SEQ ID NO: 248<10 μM SEQ ID NO: 249 <10 μM SEQ ID NO: 250 <10 μM SEQ ID NO: 251 <10 μMSEQ ID NO: 252 <10 μM SEQ ID NO: 253 <10 μM SEQ ID NO: 254 <10 μM SEQ IDNO: 255 <10 μM SEQ ID NO: 256 <10 μM SEQ ID NO: 257 <10 μM

In FIG. 1, a dose-response profile for exemplary peptides is reported.

Example 6: Phenotipic Assay in Human Differentiated TH17 Cells andViability Assay

To evaluate the effect of the peptides of the invention on the targetcells responsible for inflammation in ophtalmic or dermatologicalpathologies, we tested the ability of the peptide of SEQ ID NO: 18 toinhibit in vitro pro-inflammatory cytokines and metalloproteinasessecretion by human Th17 cells.

Human primary CD4 T-cells were differentiated into Th17 cells in vitroby culturing the cells in Th17 differentiation medium for 10 days. Indetails, purified human peripheral blood CD4 T-cells were cultured inTh-17 differentiation medium (CellXVivo Human Th17, Tocris) permanufacturer's instructions. Briefly: 96-well tissue culture plates werecoated with anti-CD3 and CD28 agonistic antibodies. Cells were countedvia trypan blue exclusion and suspended at a concentration of 1E5cells/ml in Th-17 differentiation medium. 0.2 mL of this suspension wasplated into individual wells of a 96-well plate. Cells were incubated at37° C. with 5% CO2 for 10 days, with media replaced as needed. Following10-day Cell Differentiation, the peptides synthetized in Example 1, suchas the peptide of Seq. ID No 18, were diluted to the appropriateconcentration in CGM (cell growth medium, XVivo-15, Lonza). Cell platewas centrifuged at 300×g for 5 min, and Th17 differentiation medium wasremoved.

Following differentiation, the cells were stimulated with both anactivation cocktail containing Phorbol 12-myristate 13-acetate andlonomycin in the presence of varying concentrations of test articles.

At 24 h following stimulation, culture supernatants were analyzed forexpression of IL-17, IL-6, IL-23, MMP3 and examined for viability usingthe fluorescence viability dye, Alamar blue. The CD4 cells secreted highlevels of IL-17A in response to stimulation with an activation cocktail,indicating successful differentiation into Th17 cells. Additionally,stimulated cells secreted IL-6, IL23 and MMP3.

Supernatant was removed and replaced with 200 μL of diluted peptide orvehicle (CGM+DMSO diluted as with TI concentration) in appropriategroups. Cells were incubated with peptide test items for 1 hour beforestimulation. The following stimulation solutions were generated: 10×Cell Activation Cocktail: Diluted from 500× to 10× working solution inCGM. 22 μL of appropriate stimulants were added to wells 1 hr after TIaddition. Finally, 25 μL of Alamar blue reagent (10×) was added to eachwell 20 hours after stimulant (4 hrs prior to 24 hr serum collection) todetermine cell viability. 24 hrs After Stimulant, Alamar bluefluorescence was measured per manufacturer's instructions. RemainingSupernatants were collected for subsequent multiplex protein analyses.Expression levels of IL-6, IL-17, and IL-23 as well as MMP3 in 24 hourstimulated culture supernatants was evaluated using a Luminex MultiplexPlatform on a MAGPIX instrument.

FIGS. 2 and 3 show the effect of the peptide of SEQ ID NO: 18. As can beseen, this peptide is able to significantly reduce the secretion ofIL-17A, IL-6 and IL-23 as well as the secretion of MMP3. As shown inFIG. 4 the inhibition of cytokines and metalloproteinases secretion isnot due to cell viability issues since cell treatment with the peptideof SEQ. ID N. 18 for eleven days did not impair cell viability.

Example 7: Physical-Chemical Analysis of Peptides

The physical-chemical properties of the prior art peptide HAP and ofsome peptides according to the invention (see table 8) have been testedas described below.

i. Methods

a. Isoelectric Poitn IP Log D and log P

The main physicochemical properties (Isoelectric point (IP), log D (7.4)and log P) of the peptides of Table 8 below were determined by using theSiriusT3 apparatus (Sirius Analytical Instruments Ltd., East Sussex, UK)equipped with an Ag/AgCl double junction reference pH electrode, aSirius D-PAS spectrometer and a turbidity sensing device. The pHelectrode was calibrated titrimetrically in the pH range 1.8-12.2. Anoverhead stirrer was used and a temperature probe (Peltier controller)monitored the temperature during the course of the titration. Thetitration experiments were conducted in 0.15 M KCl Ionic StrengthAdjusted solution (ISA water) under a nitrogen atmosphere at atemperature of 25±1° C. All tests were performed using standardized 0.5M KOH and 0.5 M HCl as titration reagents, for the partition coefficienttests a saturated Octanol in ISA water (5% of ISA water) was used aspartition solvent. The pKas were determined by potentiometric method bypH-metric titration. The powder (around 0.5 mg) was dissolved in 1.5 mlof ISA water and the titration was performed in triplicate in the pHrange 2.0−12.0. Each Log P were performed in triplicate by dissolvingthe powder (around 1 mg) in 1 ml of ISA water followed by pH metrictitration in three different percentages of octanol (in general 50%,60%, 70%).

b. Water Solubility and Stability

General Procedure for the Preparation of the Stock Solutions

Each lyophilized peptide was weighed in an amber vial. A calculatedvolume of phosphate buffered solution at pH 7.8 was added in order todirectly obtain 10 mM Stock Solutions. The recovered suspensions werestirred for 30 minutes by orbital shaker incubator at 37° C. Subsequentdilutions were performed to obtain the more diluted stock solutions forthe less soluble compounds.

In order to evaluate the stability and the solubility, all the stocksolutions were stored at 25° C. The title was evaluated by UHPLC-MSanalysis in comparison with the standard solutions, at 3 time points:T0, 3 days, 1 week.

The chromatographic profile was evaluated for the quantitative assaywhile the MS spectra were detected as support in the stability studies.

General Procedure for the Standard Solution Preparation

Each peptide was weighed and directly diluted at 100 μM with thephosphate buffer at pH 7.8. The solutions were stirred for 5 minutes byvortex and heated at 37° C. for 20 minutes. The obtained standardsolution was stored at 25° C. in the dark under controlled temperature.

General Procedure for the Sample Preparation

A portion of the stock solutions was collected in an amber vial anddiluted to 100 μM by phosphate buffer at pH 7.8 (dilution factor,1:100).

Instrument Method

Reversed-phase UHPLC was performed operating at a flow rate of 0.3ml/min with a Luna Omega Polar C18 column (pore size 1.6 μm, 2.1×100mm). Solvent A was 0.5% Formic Acid in water and solvent B was 0.5%Formic Acid in acetonitrile. First, samples were loaded onto the columnand washed isocratically at 10% B for 3 min. Then, a gradient of 10-95%B was run over 22 min, held at 95% for 2 min, and then decreased to 10%over 1 min and held, for the pressure stabilization, for 5 min. UVAbsorption was monitored by DAD (220-400 nm). ESI-MS ion trap was usedfor the monitoring of TIC.

ii. Results

All peptides tested showed stability at 25° C. up to 1 week since nosignificant variations were found in the chromatographic profile and inthe mass spectra.

The physchemical properties of the tested peptides are displayed inTable 8 below

TABLE 8 PEPTIDE IP Log D (7.4) logP SOLUBILITY SEQ ID NO: 1 5.14 −3.68−5.98 >10 mM SEQ ID NO: 2 5.14 −0.56 0.99 >10 mM SEQ ID NO: 3 3.90 −1.871.33 10 μM SEQ ID NO: 4 3.90 −1.32 1.88 20 μM SEQ ID NO: 5 5.19 −1.26−0.51 1 mM SEQ ID NO: 6 5.14 −3.65 −0.37 >10 mM SEQ ID NO: 7 5.19 −1.41−0.86 >10 mM SEQ ID NO: 8 5.14 −4.06 −5.12 >10 mM SEQ ID NO: 9 8.28−1.08 −0.53 >10 mM SEQ ID NO: 10 5.14 −3.66 0.09 >10 mM SEQ ID NO: 114.25 −2.92 1.05 >10 mM SEQ ID NO: 12 3.66 −5.24 1.31 10 μM SEQ ID NO: 136.06 1.15 −0.49 >10 mM SEQ ID NO: 14 8.99 −2.80 −5.40 >10 mM SEQ ID NO:15 3.51 1.88 1.66 50 μM SEQ ID NO: 16 5.19 0.24 0.13 >10 mM SEQ ID NO:17 8.05 −3.68 −0.44 >10 mM SEQ ID NO: 18 3.55 −3.79 1.89 50 μM SEQ IDNO: 41 3.54 −1.94 1.24 >10 mM SEQ ID NO: 19 12.49 −3.94 −8 100 μM SEQ IDNO: 20 11.29 −3.69 −6.79 100 μM SEQ ID NO: 21 10.6 −0.55 −4.99 >10 mMSEQ ID NO: 22 4.26 −3.18 0.4 >10 mM SEQ ID NO: 23 4.26 −2.24 1.01 50 μMSEQ ID NO: 24 5.14 2.15 2.45 <50 μM SEQ ID NO: 25 4.66 −5.64 0.4 100 μMSEQ ID NO: 26 8.13 0.148 0.69 >10 mM SEQ ID NO: 27 5.14 0.148 0.69 >10mM SEQ ID NO: 28 12.25 −6.44 −6.36 100 μM SEQ ID NO: 29 12.25 −6.44−6.39 >10 mM SEQ ID NO: 30 12.96 −3.89 −7.44 100 μM SEQ ID NO: 31 12.5−3.59 −4.9 >10 mM SEQ ID NO: 32 na −3.59 −10 <50 μM SEQ ID NO: 33 4.46−1.06 1.99 100 μM SEQ ID NO: 34 5.14 −3.38 −5.43 <50 μM SEQ ID NO: 355.14 −3.52 −5.43 >10 mM SEQ ID NO: 36 8.13 −1.24 −1.09 100 μM SEQ ID NO:37 5.14 −3.9 −6.89 >10 mM SEQ ID NO: 38 5.14 −3.46 −5.88 >10 mM SEQ IDNO: 39 4.2 −2.86 −5.36 >10 mM SEQ ID NO: 40 4.21 −3.5 −4.93 >10 mM SEQID NO: 111 8.99 −0.93 −0.38 <50 μM SEQ ID NO: 112 8.99 −7.295 −4.76 <50μM SEQ ID NO: 113 8.99 −3.472 −0.13 <50 μM SEQ ID NO: 114 8.99 −3.591−0.21 <50 μM SEQ ID NO: 115 8.99 −3.914 −5.94 <50 μM SEQ ID NO: 121 8.99−1.474 −0.51 <50 μM

As can be seen from the above, most of the peptides according to theinvention have optimal physchem properties, showing a good balancebetween solubility and lipophilicity. The mean of Log D of the peptideswas −2.64±2.06, while for most of the peptides, the solubility observedin water was very good (>10 mM). The properties observed make themparticularly suitable to be used for ophthalmic and dermatologicalapplications, since both hydrophobic and hydrophilic components arepresent in the derma and eye surface. They also explain the goodpermeability observed by peptides (see Example 8).

Example 8: Permeability Test

The peptides tested were the peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 7, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 111,SEQ ID NO: 113, SEQ ID NO: 115 and SEQ ID NO: 121. They were dissolvedin Cell Culture Grade Water (Corning, Manassas, Va., USA) in order tohave 10 mM stock solution.

i. Methods

In Vitro Model

The I-HCEC, Immortalized Human Corneal Epithelial cell line was derivedfrom Primary Human Comeal Epithelial Cells (>99% purity). These cellsare suitable for studies of human cornea in health and disease. Thecells were cultured following the protocol and using the media suggestedby Innoprot, Bizkaia, Spain (IM-Corneal Epithelial Cell Medium ref.P60131).

Immunofluorescence

Qualitative and quantitative techniques were developed to first confirmand quantify the barrier integrity before proceeding with drug testing.First, we performed immunofluorescence for a protein characteristic oftight junctions (Z0-1) (Sugrue S P et al., (1997) Exp Eye Res. January;64 (1): 11-20). The cells were seeded in coverslip coated with CollagenI 50 μg/ml (stock solution 3 mg/ml from Gibco, NY, USA) at a density of1*104 cells/cm2 and cultured for 14 DIV and post-confluency the mediawere refreshed every day. Cells were fixed in methanol at −20° C. for 10min, rinsed with phosphate buffer solution (PBS), incubated for 10 minat room temperature (RT) in PBS containing 4% bovine serum albumin (BSA;Sigma-Aldrich), and successively incubated with the primary andsecondary antibodies for 60 min each at RT. Primary antibody was goatanti-human Z0-1 (1:100, Thermo Fisher) overnight at 4° C. followed byanti-goat Alexafluor 488 (1:10000). Cells were finally counterstainedwith DAPI (0.5 mg/ml) (Sigma-Aldrich) for 5 min at room temperature,mounted with Vectashield Mounting Medium (Vector Laboratories Inc,Burlingame, Calif.) and observed at confocal laser scanning Microscope(Leica TCS SP5, Wetzlar, Germany).

MTS Assay

Cell viability was determined at 24 h using Cell Titer One Solution CellProliferation Assay (Promega Corporation Madison, Wis., USA) acolorimetric method based on3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenii)-2-(4-sulfophenyl)-2H-tetrazolium(MTS). The quantity of formazan formed, as a function of viability, wasmeasured at 492 nm using an ELISA plate reader, Infinite F200 (Tecan,Mannedorf, Swiss). The assay was performed at 50 and 100 μM (N=9). Theresults were expressed as absorbance at 492 nm.

TEER Assay

The barrier integrity of corneal epithelial cultures was verified usingmeasurement of TEER (TransEpithelial/TransEndothelial ElectricalResistance)) and permeability of paracellular permeants (e.g., mannitol,6-carboxyfluorescein) (Ronkko S et al., (2016) Drug Deliv Transl Res 6:660-675). TEER is a widely accepted quantitative technique to measureintegrity of tight junction dynamics in cell culture models ofendothelial and epithelial monolayers. TEER values are indicators of theintegrity of the cellular barriers before they are evaluated fortransport of drugs or chemicals. TEER measurements can be performed inreal-time without cell damage and generally are based on measuring osmicresistance or measuring impedance across a wide spectrum of frequencies(Snirivasan et al., 2015 J Lab Autom. 20(2): 107-126). To perform thisassay Millicell ERS-2, Electrical Resistence System by EMD Millipore(Burlington, Mass., USA), was used following manufacturer'sinstructions. The assay was performed at 50 and 100 μM (N=9). Resultswere expressed as Resistance (Q/cm2).

Permeability Test

The objective of this study was to evaluate the apparent permeabilitycoefficient (Papp) value of the peptides across stratified human cornealepithelial cells. All peptides were tested at 50 μM in duplicate.

In particular, human corneal epithelial cells (hCEPIC) were stratifiedon Transwell cell culture inserts (Corning, N.Y., USA). The permeationexperiments were performed taking samples from receiver chambers at 5,30, 60 minutes time points and from donor chambers at the same timepoints. Each aliquot collected was analyzed by HPLC instrument (Knauer)equipped with C18 Vertex Plus Column (Knauer) thermostaticallycontrolled at 40° C. The rate (apparent permeability coefficient, Papp)of the study compounds was calculated and compared to the Papp values ofthe low and high permeability markers, 6-carboxyfluorescein andRhodamine B, respectively.

The apparent permeability coefficient (Papp) was calculated according toEquation:Papp=(I/A*C0)(dM/dt)

where dM/dt is the flux (nanomoles per second) across the cell layers;

A (square centimeters) is the exposed surface area of the insertmembrane;

C0 is the initial drug concentration (micromolar) in the donorcompartment at t=0 (Xiang et al., Drug Metab Dispos. 2009 May;37(5):992-8.

Statistics

All groups (studied compound and rhodamine) were compared to lowpermeability control. All the other experiments were expressed as themeant S.E. and the significance were calculated by Student's t testversus control cells. Difference were considered to be statisticallysignificant (p<0.05) as determined by Student's t test.

ii. Results

MTS and TEER Assays

In our experimental conditions, the peptides considered did notsignificantly affect cell viability or TEER at 50 μM and 100 μM testconcentration.

Permeability Test

The results obtained for each peptide are shown in Table 9 below.

TABLE 9 Permeability Peptide N/Name Sequence (10⁻⁶ cm/sec) SEQ ID NO: 1IHVTIPADLWDWINK 19.333 SEQ ID NO: 7 IHVTIPADLWEWINK 24.252 SEQ ID NO: 11IHVTIPADLWDWIEK 28.020 SEQ ID NO: 2 VHVTIPADLWDWINK 28.862 SEQ ID NO: 14IHVTIPADLWDWVRR 28.873 SEQ ID NO: 38 VPGAGVPGAGIHVTI 30.417 PADLWDWINKSEQ ID NO: 121 VHVTIPADLWEWFRR 47.083 SEQ ID NO: 115 VHVTIPQDLWEWVRR68.800 SEQ ID NO: 113 VHVQIPADLWEWVRR 76.797 SEQ ID NO: 111VHVTIPAELWEWVRR 83.698 Rhodamine B 51.26 6-carboxyfluorescein 0.61

To verify that the HCE cell culture was useful in screening thepeptides, the permeability of a lipophilic transcellular markerrhodamine B was determined. As expected, rhodamine B exhibited highpermeability of the HCE cell culture (51.26±5.44 10⁻⁶ cm/sec). Then, thepermeability of 6-carboxyfluorescein was assayed and in agree with theliterature (0.61±0.33 10⁻⁶ cm/sec). This marker has a very lowlipophilicity, and it permeates biological membranes only through theintercellular space.

The tested peptides showed a very good permeability towards theImmortalized Human Corneal Epithelial compared to high permeabilitystandard, Rhodamine B. Obviously, the HCE permeability model does notinclude stroma or endothelium, but these layers are not criticalbarriers to corneal drug absorption. We found peptides with a goodpermeability (SEQ ID NO: 2) and peptides with a higher permeabilitycompared to Rhodamine B (SEQ ID NO: 115, SEQ ID NO: 113 and SEQ ID NO:111).

Example 9: Peptide-IL-17A Affinity Test

The peptide of the prior art HAP (SEQ ID NO:1) and peptide of SEQ IDNO:2 were tested for affinity for mIL-17A, as described below.

a. Immobilization of mIL-17A

For the immobilization of mouse IL-17A protein, amine coupling chemistrywas used. On the senorchip surface the dextran matrix was firstactivated with a mixture of 1-ethyl-3-carbodiimide (EDC) andN-hydroxysuccinimide (NHS) in order to obtain reactive succinimidoesters. 10 ug/ml of IL17 was diluted in 10 mM acetate buffer at pH=5.0and then the ligand passed over the surface to allow the esters to reactspontaneously with uncharged amino groups, in this way the ligand iscovalently linked to dextran. Covalent immobilization of mIL17 resultsin stable attachment of 3000 RU of ligand to the surface.

b. Characterization of the Peptides/IL17 Interaction

The peptides of SEQ ID NO: 1 (HAP) and SEQ ID NO: 2 were analyzed fortheir ability to bind the immobilized mIL-17A. The molecules werediluted at different concentrations (from 62.5 to 1000 nM) in HBS-EPbuffer and injected on the sensorchip for 4 minutes and allowed todissociate for 5 minutes. After each run, the sensorchip was regeneratedby 2 M NaCl injection. Based on the sensorgrams (FIG. 1, panel A) andusing the BIAevaluation software, the Scatchard analysis of the RUvalues at equilibrium allowed to determine an affinity constant as afunction of the analyte concentration in solution (FIG. 6, panel B). Theresults obtained are shown in Table 10 below

TABLE 10 Compound Kdea (nM) SEQ ID NO: 1 3700 SEQ ID NO: 2 130

As shown in Table 8, the peptide of SEQ ID NO: 2 binds to IL-17A with avalue of affinity equal to 130 nM, while peptide of SEQ ID NO: 1 bindsto IL17-A with a very low affinity value (3700 nM).

In view of the results obtained in Example 8 and 9, the modification ofthe Isoleucin in position 1 in the HAP peptide of the prior art with aValine, as in the peptide of SEQ ID NO: 2, leads to a strikingdifference regarding the physchemical properties, permeability andaffinity for the ligand.

We summarize herein below the properties of the two peptides:

TABLE 11 SEQ ID NO: 1 Property (HAP) SEQ ID NO: 2 SequenceIHVTIPADLWDWINK VHVTIPADLWDWINK logP −5.98 0.99 logD (7.4) −3.68 −0.56Permeability 19.3 28.86 (Papp ± SE) SPR Kdeq (nM) 3700 130

Table 11 shows the comparison data between peptide SEQ ID NO: 1 and SEQID NO: 2. We found great differences in term of log P and log D (6-logand 3-log difference, respectively): this explained the higher permeableobserved with SEQ ID NO: 2 compared to SEQ ID NO: 1 (28.8 vs 19.3). TheKd (nM) measured in the Surface plasmon resonance (SPR) assay performedon mouse IL-17A, showed that SEQ ID NO: 2 was 28-fold more affine thanSEQ ID NO: 1.

Furthermore, molecular dynamics simulations studies were also carriedout, which indicate that Valine (SEQ ID NO: 2) instead of the Isoleucine(HAP) greatly stabilizes the folded structure: SEQ ID NO: 2 waspredicted to be 80% more stable.

Example 10: In Vitro Evaluation of Expression of Inflammatory Cytokines

The peptide HAP (SEQ ID NO:1) and the peptide of SEQ ID NO:2 were testedin vitro for evaluation of their direct influence on expression ofinflammatory cytokines in the cornea. The tests were conducted onImmortalized Human Corneal Epithelial cell line (I-HCEC) (Innoprot,Bizkaia, Spain, Ref. P10871). This is derived from Primary Human CornealEpithelial Cells (>99% purity) and is suitable for studies of humancornea in health and disease. The cells were cultured following theprotocol and using the media suggested by Innoprot (IM-CornealEpithelial Cell Medium ref. P60131). The cell medium was supplementedwith 5% Fetal Bovine Serum (FBS).

Cells were seeded at 320000 cells/well 6 plate and 15000 cells/well 96plate. When the cells reached 80% of confluence were treated with SEQ IDNO: 1 and SEQ ID NO: 2 compounds in dose-curve (0.01-500 μM) for 24hours.

Total RNA was isolated from 6 wells plate using Quick-Start RNeasy MiniKit (Qiagen, cat. No 74104) according to the manufacturer'sinstructions. Retro-transcription was performed using SuperScript Vilo(ref. 11755050, Life technologies) and real-time PCR using TaqManprotocol (ref. 4444557, Applied Biosystem) adapted for CFX96 Real-TimeSystem. The experiments were performed three times, each time induplicate. The used probes from Thermo Fisher Scientific are: humanCXCL8 (Hs00174103_m1), human IL6 (Hs00174131_m1), human TNFα(Hs99999043_m1). As housekeeping gene was used GAPDH (probeHs02758991_g1). IL-8, IL-6 and TNF-α expression in I-HCEC was evaluatedafter treatment for 24 hours with different concentration of the peptideof SEQ ID NO: 1 or SEQ ID NO: 2 (0.01, 0.1, 1, 10, 100, 500 μM).Untreated I-HCEC were used as negative control (Vehicle).

The results show that the compound SEQ ID NO: 2 does not change theexpression of any of the measured inflammatory cytokines (FIGS. 8A, 9Aand 10A), while the treatments with 500 μM of the compound SEQ ID NO: 1significantly induce the expression of the two inflammatory cytokinesIL-6 and TNFα (FIGS. 9B and 10B).

The data obtained demonstrate that peptide of SEQ ID NO: 1 has a directpro-inflammatory activity and induces significantly the expression ofIL-6 and TNF-α inflammatory cytokines by HCEC, with an increase of 10and 8 fold, respectively. On the contrary, the peptide of SEQ ID NO: 2does not show any effect on the expression of cytokines.

In view of the above, it can be concluded that the peptide of SEQ ID NO:2 surprisingly shows significantly better tolerability and lack oftoxicity compared to the peptide HAP of SEQ ID NO: 1.

The invention claimed is:
 1. A peptide that specifically inhibits IL-17Abinding to ILRA, comprising the amino acid sequence of Formula (I),wherein Formula (I) comprises: (I) (SEQ ID NO: 258)X₁-X₂-X₃-X₄ X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅

wherein in Formula (I): (a) X₁ is I, V or L; X₂ is H, M, R, K or E; X₃is V, F or I; X₄ is T, Q, S, Y or N; X₅ is I, F or V; X₆ is P; X₇ is A,Q, or L; X₈ is D, E, or Q; X₉ is L, W, F, V or I; X₁₀ is W, Y or F; X₁₁is D, E or N; X₁₂ is W or F; X₁₃ is I, V, F or L; X₁₄ is N, R, Q or E;X₁₅ is K, R, H or E; with the proviso that said sequence is notIHVTIPADLWDWINK (SEQ ID NO:1); or (b) X₁ is I or V, X₂ is H, M or R, X₃is V or F; X₄ is T or Q; X₅ is I, F or V; X₆ is P or G; X₇ is A or Q; X₈is D or E; X₉ is L; X₁₀ is W or Y; X₁₁ is D or E; X₁₂ is W; X₁₃ is I orV; X₁₄ is N, R or E; X₁₅ is K, R or E; with the proviso that saidsequence is not IHVTIPADLWDWINK (SEQ ID NO: 1) or (c) X₁ is I or V; X₂is H or M, X₃ is V; X₄ is T; X₅ is I; X₆ is P; X₇ is A; X₈ is D; X₉ isL, W, F, V or I; X₁₀ is W or Y; X₁₁ is D or E; X₁₂ is W; X₁₃ is I or V;X₁₄ is N, R or E; X₁₅ is K, R or E; with the proviso that said sequenceis not IHVTIPADLWDWINK (SEQ ID NO: 1), HVTIPADLWDWIN (SEQ ID NO:259),IHVTIPADLWDWI (SEQ ID NO:260) or IHVTIPADLWDW (SEQ ID NO:261).
 2. Thepeptide of claim 1, wherein in formula (I) X₁ is V.
 3. The peptide ofclaim 1, having an amino acid sequence selected from SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 42, SEQ ID SEQ ID NO: 111, SEQ ID NO: 112, SEQ IDNO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118,SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ IDNO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ IDNO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145,SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ IDNO: 150, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155,SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ IDNO: 171, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177,SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ IDNO: 193, SEQ ID NO: 198, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207,SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226,SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ IDNO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235,SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ IDNO: 240, SEQ ID NO: 241, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 249,SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 254, and SEQ ID NO:
 256. 4.The peptide of claim 1, further comprising sequence (A)_(m) at the C-and/or N-terminal of the peptide, wherein A is an amino acid selectedfrom the group consisting of R, K, G, E, Q and A, and m is an integerbetween 1 and
 10. 5. A peptide as claimed in claim 4, having sequence ofSEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:43.
 6. The peptide of claim 1, wherein a protective cap group is boundat the C- and/or N-terminal, wherein the protective cap group isselected from the group consisting of amides, aldehydes, esters,p-Nitroanilide, 7-Amino-4-methylcoumarin and the protective group capbound to a N-terminal being selected from acetyl, formyl, pyroglutamyl,fatty acids, urea, carbamate sulfonamide, and alkylamine.
 7. A dimerformed by two peptides, wherein each peptide is a peptide of claim
 1. 8.The dimer of claim 7, wherein the peptides are linked by a polyethylenespacer.
 9. A bioconjugate comprising the peptide of claim 1 and abiomolecule, wherein the biomolecule is bound to the N- and/orC-terminal of the peptide.
 10. The bioconjugate of claim 9, wherein thebiomolecule is selected from the group consisting of capric acid,capronic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid,alginic acid, chitin, (GaINAc)2, Ga1-alpha1,3-GaINAc and trigalacturonicacid.
 11. A pharmaceutical composition comprising the peptide of claim 1and at least one pharmaceutically acceptable excipient.
 12. A method oftreating a subject having an inflammatory and autoimmune diseasecomprising administering to the subject an effective amount of thepeptide of claim
 1. 13. The method of claim 12, wherein the inflammatoryand autoimmune disease is selected from the group consisting ofrheumatoid arthritis, multiple sclerosis, Crohn's disease, systemiclupus erythematosus, asthma, Behçet's disease, hyper IgE syndrome,ankylosing spondylitis, psoriasis, psoriatic arthritis, rheumatoidarthritis, keratoconjunctivitis sicca, vernal keratoconjunctivitis,stromal herpetic keratitis, corneal allograft rejection, cornealinfections, herpes virus or Pseudomonas aeruginosa keratitis and dry eyedisease, preferably psoriasis and dry eye disease.
 14. A method ofproducing the peptide of claim 1, comprising (a) synthesizing thepeptide; and (b) purifying the peptide.
 15. The peptide of claim 1,comprising an amino acid sequence of SEQ ID NO:
 2. 16. The peptide ofclaim 1, having a 15 amino acid sequence.